Encapsulated apomorphine compositions

Abstract

The invention relates to a method of treating a neurological disorder in which an increase in dopaminergic turnover is beneficial, the method comprising intranasal administration of an encapsulated apomorphine composition to a subject in need thereof.

Description

[0001] ENCAPSULATED APOMORPHINE COMPOSITIONS

[0002] 1. FIELD OF THE INVENTION

[0003] The present invention relates to the use of encapsulated apomorphine compositions for nasal administration, to be used in the treatment of neurological disease, such as Parkinson's disease.

[0004] 2. BACKGROUND TO THE INVENTION

[0005] Neurodegenerative diseases are becoming more prevalent as populations age.

[0006] Parkinson's disease is one of the most severe progressive neurodegenerative disorders, having a mortifying effect on the health of millions of people around the globe.

[0007] In Parkinson's disease, the neural cells producing dopamine in the substantia nigra of the brain die out. Dopamine is a chemical neurotransmitter that is utilised by brain cells to transmit impulses that control or modulate peripheral muscle movement. The degeneration of dopamine-containing neurons reduces the amount of dopamine in the brain. This process is thought to disturb nerve cell functions such that impulses are not transmitted properly, resulting in a loss of muscle control and function. This leads to symptoms like hypokinesia, rigidity, bradykinesia, and rest tremor.

[0008] Currently, there is no cure for Parkinson's disease. Treatments are typically aimed at alleviating Parkinson's disease symptoms, primarily by replacing the dopamine with either (levo)-3,4-dihydroxyphenylalanine (L-DOPA), which is metabolised to dopamine, or by administering chemical agents that stimulate the dopamine receptors. These receptors fall into two broad classes, Dl-type and D2-type receptors. The former is divided into DI and D5 receptors, while the D2 receptor family consists of D2, D3 and D4 receptors.

[0009] Delivering drugs to the brain for treating Parkinson's disease is very challenging. The blood-brain barrier acts as a highly selective semi-permeable barrier, preventing most drugs from reaching the brain. Conventional drug delivery systems used for Parkinson's disease do not readily cross the blood-brain barrier leading to severe side-effects. Apomorphine is (6aR)-6-methyl-5, 6,6a, 7-tetrahydro-4H-dibenzo[de, g]quinoline- 10,11-diol. It has the chemical formula:

[0010]

[0011] Apomorphine is a very potent dopamine agonist which acts on both DI and D2 receptors. It is very lipophilic and so readily crosses the blood-brain barrier.

[0012] Unfortunately, it has poor oral absorption and a short half-life.

[0013] Current formulations for clinical use include subcutaneous injection, continuous subcutaneous infusion, sublingual films and pulmonary inhalation as a dry powder.

[0014] Intranasal administration of a dry powder has been trialled but discontinued due to toxicity and efficacy issues.

[0015] In all of these delivery forms, apomorphine has a rapid onset of up to 15 minutes but a short elimination half-life of 30 minutes, providing a clinical effect duration of only about 30-60 minutes. The exception is continuous subcutaneous infusion, which is highly invasive.

[0016] Animal studies indicated that transdermal delivery or delivery via implants may provide useful modes of administration of apomorphine. However, when the delivery of apomorphine from implants was studied in monkeys it was found that in most cases, the animals had to be treated with the immunosuppressant dexamethasone to prevent local irritation and other complications following implantation surgery. Transdermal delivery of apomorphine has also been associated with local skin irritation and colouration.

[0017] There is therefore still a need for a means of administering apomorphine that can overcome the problems associated with its delivery and half-life, or at least to provide the public with a useful choice.

[0018] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. 3. SUMMARY OF THE INVENTION

[0019] The invention relates to a method of treating a neurological disorder in which an increase in dopaminergic turnover is beneficial, the method comprising intranasal administration of an encapsulated apomorphine composition to a subject in need thereof.

[0020] In one embodiment the method provides a therapeutic improvement in motor function in the subject lasting at least 2-hours in duration.

[0021] In one embodiment the encapsulated apomorphine composition comprises one or more encapsulated apomorphine constructs selected from the group comprising liposomal apomorphine, polymeric apomorphine nanoparticles, lipid apomorphine nanoparticles, protein-encapsulated apomorphine, protein-bound apomorphine, and mesoporous nanoparticles of apomorphine.

[0022] In one embodiment the encapsulated apomorphine composition comprises liposomal apomorphine.

[0023] In one embodiment the ratio of apomorphine in the intraliposomal compartment to the total lipid in the lipid membrane is about 0.05: 1 to about 5:1 (mol / mol) and / or about 0.05:1 to about 5:1 (wt / wt).

[0024] In one embodiment the disorder is Parkinson's disease.

[0025] 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention will now be described by way of example only and with reference to the drawings in which:

[0027] Figure 1 is a cryo-transmission electron micrograph showing the morphology of liposomal apomorphine.

[0028] Figure 2 is a graph showing the percentage of apomorphine released from apomorphine sulfate and apomorphine oxalate liposomes when incubated with 30% blood plasma or phosphate buffered saline for 30 minutes.

[0029] Figure 3 is a graph showing the release of apomorphine and calcein from apomorphine-calcein liposomes. (A) Raw fluorescence intensity of apomorphine when incubated with 30% blood plasma or phosphate buffered saline (PBS) for 2, 15, and 30 minutes. (B) Percentage apomorphine release from apomorphine-calcein liposomes incubated with PBS and 30% blood plasma for 30 minutes. (C) Raw fluorescence intensity of calcein when incubated with 30% blood plasma or phosphate buffered saline (PBS) for 2, 15, and 30 minutes. (D) Percentage calcein release from apomorphine-calcein liposomes incubated with PBS and 30% blood plasma for 30 minutes.

[0030] Figure 4 is a graph showing that liposomal apomorphine provides a greater duration of action over apomorphine hydrochloride when administered intranasally. A difference in time to fall can be seen between liposomal apomorphine and apomorphine at 2 hours where liposomal encapsulation has extended the therapeutic duration. Error bars represent standard error of the mean (n=7 / 8).

[0031] 5. DETAILED DESCRIPTION OF THE INVENTION

[0032] 5.1 Definitions and abbreviations

[0033] As used herein the term "comprising" means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner. The term "about" as used herein means a reasonable amount of deviation of the modified term such that the end result is not significantly changed. For example, when applied to a value, the term should be construed as including a deviation of+ / - 5% of the value.

[0034] The term "encapsulated apomorphine composition" refer to a composition comprising one or more encapsulated apomorphine constructs. An encapsulated apomorphine construct is apomorphine is a form in which the apomorphine compound is in some way surrounded by, enclosed within, or bound to a protective coating or matrix. This coating or matrix may consist of various materials, such as polymers, lipids, proteins, or other suitable substances, designed to control the release, protect the stability, or enhance the bioavailability of the active pharmaceutical ingredient, apomorphine. Examples of encapsulated apomorphine constructs include but are not limited to, liposomal apomorphine, polymeric apomorphine nanoparticles, lipid apomorphine nanoparticles, protein-encapsulated apomorphine, protein-bound apomorphine, and mesoporous nanoparticles of apomorphine.

[0035] The term "liposome" refers to a small artificial vesicle, spherical in shape, having at least one lipid bilayer. The "liposomal membrane" is the outer lipid bilayer of a liposome. The term "intraliposomal compartment" refers to the volume sequestered inside the liposomal membrane. The lipid bilayer is an arrangement of amphiphilic lipid molecules characterised by a hydrophilic moiety and a hydrophobic moiety, arranged in two-dimensional sheets in the which the hydrophobic moieties are oriented inwards while the hydrophilic moieties are oriented outward. Liposomes can be unilamellar (with one lipid bilayer) or multilamellar (with more than one lipid bilayer).

[0036] Liposomes generally have a spherical shape but reference to a liposomal diameter should not be taken to imply that the liposomes are completely spherical. References to liposomes as having a particular size should also not be taken to mean that every liposome with a population of liposomes has that size.

[0037] The term "liposomal apomorphine" refers to a liposome comprising lipids and apomorphine or a salt thereof, the lipids forming a liposomal membrane enclosing an aqueous intraliposomal compartment containing the apomorphine or salt thereof.

[0038] The term "polymeric apomorphine nanoparticle" refers to nanoscale particles in which apomorphine is encapsulated within or conjugated to a polymeric material. These nanoparticles may typically range from 1 to 1000 nanometres in size and are engineered to serve as a delivery system for apomorphine. The polymeric matrix provides protection for apomorphine, allowing for controlled release, improved stability, and enhanced targeting within the body. The encapsulation within the polymeric nanoparticle is designed to optimise the therapeutic efficacy of apomorphine by improving its bioavailability, minimising degradation, prolonging release and / or enabling site-specific delivery.

[0039] The term "lipid apomorphine nanoparticle" refers to nanoscale particles in which apomorphine, the active pharmaceutical ingredient, is encapsulated within or associated with a lipid-based structure. These nanoparticles may typically range from 1 to 1000 nanometres in size and are engineered as a delivery system for apomorphine. The lipid matrix which may include phospholipids, cholesterol, sphingolipids, synthetic lipids, polymer-conjugated lipids, or other lipid-based materials, provides a protective environment for apomorphine, facilitating controlled release, improved stability, and targeted delivery. The lipid encapsulation enhances the bioactivity of apomorphine, protects it from degradation, and enables efficient interaction with biological membranes, optimising the therapeutic efficacy of apomorphine.

[0040] The term "protein-encapsulated" or "protein-bound" apomorphine may be used interchangeably and refer to a pharmaceutical formulation in which apomorphine is enclosed, immobilised, adsorbed, or chemically bonded to a protein structure. This encapsulation or binding can enhance the stability, bioavailability, targeted delivery, or controlled release of apomorphine from the formulation. The protein may serve as a carrier, stabiliser or mediator for apomorphine interaction with a biological system, improving its therapeutic efficacy and stability while potentially reducing side effects. The protein-drug interaction can be achieved through various mechanisms, including covalent boding, electrostatic interactions, hydrophobic forces or through the formation of protein-based nanostructures. This method allows for improved pharmacokinetics, site-specific delivery, and prolonger circulation time and therapeutic efficacy in vivo. The term "mesoporous nanoparticles of apomorphine" refers to nanoscale particles characterised by a mesoporous structure, typically with pore sizes ranging from 2 to 50 nanometres, in which apomorphine is encapsulated or adsorbed, for example where apomorphine is adsorbed in a mesoporous silica nanoparticle. These mesoporous nanoparticles are engineered to serve as a delivery system for apomorphine, providing a large surface area and controlled pore environment that facilitate the loading, release, and stabilisation of the drug. The mesoporous structure allows for controlled release kinetics, enhanced bioavailability and protection of apomorphine from environmental degradation, thereby optimising its therapeutic efficacy. Mesoporous nanoparticles may be fabricated from silica, silicon dioxide, carbon, polymers, metals and metal oxides (including for example titanium dioxide, iron oxide and zinc oxide), metal-organic frameworks (MOFs), and / or phosphates (including for example calcium phosphate). The design of these nanoparticles ensures efficient drug delivery, particularly in targeted or sustained-release applications.

[0041] The terms "treating", "treat" and "treatment" refer to the amelioration of the symptoms associated with the neurological disorder in which an increase in dopaminergic turnover is beneficial. "Amelioration" is used in a broad sense to refer to at least a reduction in the magnitude of a parameter associated with the disorder. For non-limiting example, a reduction in dyskinesia, a reduction in off-periods or off-time, and an improvement in overall motor and non-motor function, such as that measured by the Unified Parkinson's Disease Rating Score (UPDRS or MPD-UPDRS).

[0042] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0043] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. In the disclosure and the claims, "and / or" means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.

[0044] 5.2 The method of the invention

[0045] The method of the invention is based on the surprising discovery that intranasal administration of encapsulated apomorphine, in particular, liposomal apomorphine, can overcome issues associated with delivery of apomorphine to the brain.

[0046] Liposomes are self-assembled (phospho)lipid-based vesicles that form a bilayer or concentric series of multiple bilayers enclosing a central aqueous intraliposomal compartment. They range in size from 30 nm to the micrometer scale, with the phospholipid bilayer being about 4-5 nm thick. Liposomes are considered to be promising drug delivery carriers because they are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes and transport their load across biological membranes.

[0047] However, many prerequisites must be met in order to achieve clinically viable liposome formulations that can be administered to a patient. These include achieving a sufficient level of drug loading; maintaining the drug in the liposomes when exposed to the blood plasma or patient tissues; releasing the drug at the target site at a rate and level that is sufficient to result in the desired therapeutic efficacy; and achieving a pharmaceutically accepted product in terms of shelf-life stability.

[0048] For in vivo and clinical applications, liposomes should ideally contain a maximal amount of therapeutic agent per volume of liposome, while maintaining minimal drug leakage. The liposomal apomorphine compositions of the invention go some way towards achieving these targets, when administered intranasally.

[0049] Liposomes have previously been used for delivery of active agents against neurodegenerative diseases such as Alzheimer's and Parkinson' Disease. (Navarro, 2011) Unfortunately, as shown by the inventor in Example 2, liposomal apomorphine is unstable in blood plasma. Approximately 80-100% of the encapsulated apomorphine was released from the liposome when incubated for 30 minutes in buffered sheep blood plasma (see Figures 2 and 3). This means that liposomal apomorphine is unlikely to meet the prerequisites needed for clinical use, when administered intravenously.

[0050] Surprisingly, the inventor found that intranasal administration of liposomal apomorphine did not suffer the same disadvantages. As set out in Example 4, intranasal administration of both apomorphine and liposomal apomorphine extended the mean latency-to-fall time relative to the vehicle control, but liposomal apomorphine extended the time further. The improvement can be observed after 2 hours, indicating that the liposomal apomorphine is stable when administered intranasally. The results suggest an extension of dopaminergic agonist activity when liposomal apomorphine is administered intranasally.

[0051] Accordingly, in one aspect the invention provides a method of treating a neurological disorder in which an increase in dopaminergic turnover would be beneficial, the method comprising intranasal administration of an encapsulated apomorphine composition to a subject in need thereof.

[0052] The invention also provides an encapsulated apomorphine composition for use in a method of treating a neurological disorder in which an increase in dopaminergic turnover would be beneficial, wherein the composition is formulated for intranasal administration. Accordingly, in one aspect the invention provides a method of treating a neurological disorder in which an increase in dopaminergic turnover would be beneficial, the method comprising intranasal administration of an encapsulated apomorphine composition to a subject in need thereof.

[0053] The invention also provides an encapsulated apomorphine composition for use in a method of treating a neurological disorder in which an increase in dopaminergic turnover would be beneficial, wherein the composition is formulated for intranasal administration. The invention also provides a use of an encapsulated apomorphine composition in the manufacture of a medicament for treating a neurological disorder in which an increase in dopaminergic turnover would be beneficial, wherein the medicament is formulated for intranasal administration.

[0054] In one embodiment the encapsulated apomorphine composition comprises one or more encapsulated apomorphine constructs selected from the group comprising liposomal apomorphine, polymeric apomorphine nanoparticles, lipid apomorphine nanoparticles, protein-encapsulated apomorphine, protein-bound apomorphine, and mesoporous nanoparticles of apomorphine.

[0055] In one embodiment the encapsulated apomorphine composition comprises liposomal apomorphine.

[0056] Intranasal administration of apomorphine has been tried unsuccessfully by many researchers. Both dry-powder and aqueous formulations of apomorphine have been developed for intranasal use. Unfortunately, the formulations used suffered from the same short duration of efficacy (30-60 minutes) as formulations delivered by other methods. Adverse reactions including nasal irritation were also observed. (Dewey R. , Maraganore, Ahlskog, & Matsumoto, 1996) (Kapoor, 1990) (Dewey R. , Maraganore, Ahlskog, & Matsumoto, 1998)

[0057] The present invention overcomes these problems by encapsulation of the apomorphine. In a preferred embodiment of the method, the encapsulated apomorphine is liposomal apomorphine. Liposomal apomorphine can be prepared using any method known in the art.

[0058] In one embodiment the intraliposomal compartment of the liposomal apomorphine comprises solid or semi-solid apomorphine salt, preferably apomorphine sulfate. In one embodiment the solid or semi-solid apomorphine salt takes up about 10 to about 60% of the volume of the intraliposomal compartment. In another embodiment, the solid or semi-solid apomorphine salt takes up about 20 to about 50% of the volume of the intraliposomal compartment. In yet another embodiment, the solid or semi-solid apomorphine salt takes up about 30 to about 40% of the volume of the intraliposomal compartment.

[0059] In one embodiment the liposomes are about 50 to 400 nm size, preferably about 50 to 330 nm, more preferably about 50 to 250 nm, and most preferably, about 50 to 150 nm size.

[0060] The lipid membrane of the liposomes comprises at least one liposome-forming lipid. Liposome-forming lipids include but are not limited to glycerophospholipids and sphingomyelins. Glycerophospholipids are lipids with a glycerol backbone where at least one, preferably two, of the hydroxyl groups of the head group are substituted by one or two of an acyl, alkyl or alkenyl chain, a phosphate group or combination of any of these and / or derivatives of the same. Glycerophospholipids may contain a chemically reactive group such as an amine, acid, ester, aldehyde or alcohol at the head group, thereby providing the lipid with a polar moiety. Examples of glycerophospholipids include but are not limited phosphatidylcholine (lecithin), phosphatidylglycerol, phosphatidylcholine, phosphatidyl ethanolamine, phosphatidylserine, phosphatidylinositol and hydrogenated glycerophospholipids such as hydrogenated soybean phosphatidyl choline (HSPC).

[0061] The sphingomyelins consist of a ceramide unit with a phosphorylcholine moiety attached at position 1 and therefore in fact are N-acyl sphingosines. The phosphorylcholine moiety is hydrophilic. The term "sphingomyelin" as used herein, includes dihydrosphingomyelin (DHSM).

[0062] DHSM for use in the methods of the invention can be obtained from natural or synthetic sources. Naturally sourced DHSM includes but is not limited to egg yolk or brain derived DHSM. Synthetic DHSM can be obtained by hydrogenating natural sphingomyelin, such as chicken egg or brain derived sphingomyelin. Alternatively, totally synthetic methods of preparing dihydrosphingomyelin can obtain 98% or more of the compound having a long-chain alkyl group having 16 carbon atoms and a long-chain alkyl group having 18 carbon atoms.

[0063] In the liposome-forming lipids the acyl, alkyl or alkenyl chain is typically between 14 to about 24 carbon atoms in length, and has varying degrees of saturation being fully, partially or non-hydrogenated naturally occurring lipids, semi-synthetic or fully synthetic lipids. The level of saturation may affect the rigidity of the liposome thus formed.

[0064] Typically, lipids with saturated chains are more rigid than lipids of same chain length in which there are un-saturated chains, especially having cis double bonds.

[0065] In one embodiment, the lipid membrane comprises a single type or a combination of liposome-forming lipids. In one embodiment, the liposome-forming lipid is a phospholipid such as a glycerophospholipid or sphingosine. When the liposome-forming lipid is a phospholipid, the amount thereof in the liposome / Aposome can be determined as organic phosphorous by the modified Bartlett method (Shmeeda, Even-Chen, Honen, Cohen, & Weintrau, 2003).

[0066] In one embodiment, the liposome-forming lipid is a choline-type phospholipid such as diacylglycero-phosphocholine (the acyl, alkyl or alkenyl chain being as defined above). In one embodiment the liposome-forming lipid is di-lauroyl-sn-glycero-2-phosphocholine (DLPC). In one embodiment the liposome-forming lipid is l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In one embodiment the liposome-forming lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In one embodiment the liposomeforming lipid is l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In one embodiment the liposome-forming lipid is l,2-diheptadecanoyl-sn-glycero-3-phosphocholine. In one embodiment the liposome-forming lipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment the liposome-forming lipid is 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine. In one embodiment the liposome-forming lipid is 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC). In one embodiment the liposomeforming lipid is l,2-dihenarachidoyl-sn-glycero-3-phosphocholine. In one embodiment the liposome-forming lipid is l,2-dibehenoyl-sn-glycero-3-phosphocholine 1,2-ditricosanoyl-sn-glycero-3-phosphocholine. In one embodiment the liposome-forming lipid is l,2-dilignoceroyl-sn-glycero-3-phosphocholine. In one embodiment the liposomeforming lipid is l-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine. In one embodiment the liposome-forming lipid is l-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC). In one embodiment the liposome-forming lipid is l-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC). In one embodiment the liposome-forming lipid is 1,2- di-oleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment the liposomeforming lipid comprises at least hydrogenated soy phosphatidylcholine (HSPC). In addition to the at least one liposome-forming lipid, the lipid membrane typically includes other lipophilic components.

[0067] In one embodiment the lipid membrane also comprises sphingomyelin. In one embodiment the lipid membrane comprises DHSM. In one embodiment the DHSM has two long chain alkyl groups having 16 carbon atoms or a long-chain alkyl group having 16 carbon atoms and a long chain alkyl group having 18 carbon atoms or a long chain alkyl group having 16 carbon atoms and a long chain alkyl group having 20 to 24 carbon atoms. Preferably, the DHSM contains a long-chain alkyl group having 16 carbon atoms and a long chain alkyl group having 18 carbon atoms.

[0068] In one embodiment the lipid membrane also comprises a sterol, such as cholesterol. In one embodiment the lipid membrane also comprises a lipopolymer. Lipopolymers comprise lipids modified at their head group with a polymer moiety, such as polyethylene glycol (PEG), having a molecular weight equal or above 750 Da. The head group may be polar or apolar, to which a large (>750 Da) flexible hydrophilic polymer is attached. The attachment of the hydrophilic polymer head group to the lipid region may be a covalent or non-covalent attachment, however, it is preferably via the formation of a covalent bond (optionally via a linker).

[0069] The lipids modified into lipopolymers may be neutral, negatively charged, as well positively charged, i.e. there is no restriction to a specific (or no) charge. For example the neutral distearoyl glycerol and the negatively charged distearoyl phosphatidylethanolamine can both be covalently attached to methoxy poly(ethylene glycol) (mPEG or PEG) of Mw 750, 2000, 5000, or 12000 (Priev, A., et al., 2002) (O, 2005) (Lasic, 1992).

[0070] The most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually, distearylphosphatidylethanolamine (DSPE). A specific family of lipopolymers employed in the liposomal apomorphine includes methoxy PEG-DSPE (with different lengths of PEG chains) in which the PEG polymer is linked to the DSPE primary amino group via a carbamate linkage. The PEG moiety preferably has a molecular weight of the head group from about 750 Da to about 20,000 Da. More preferably, the molecular weight of the head group is from about 750 Da to about 12,000 Da and most preferably between about 1,000 Da to about 5,000 Da. One specific methoxy PEG-DSPE employed herein is that wherein PEG has a molecular weight of 2000 Da, designated herein 2000PEG-DSPE, DSPE-PEG2000 or 2kPEG-DSPE (Lasic, 1992). In one embodiment the lipid membrane of the liposomal apomorphine comprises about 1 to about 10 mol% lipopolymer. In one embodiment the lipid membrane comprises at least about 1, 2, 3, 4, 5, 6, 7, 8 or 9 to about 10 mol% lipopolymer.

[0071] In one embodiment the lipid membrane of the liposomal apomorphine comprises the liposome-forming lipid (which may be one or a combination of such lipids), one or more sterols and one or more lipopolymers. The mole ratio between these three components may vary. In one embodiment the lipid membrane comprises hydrogenated soybean phosphatidylcholine (HSPC), a lipopolymer of 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (2kPEG-DSPE) and cholesterol.

[0072] In one embodiment the lipid membrane comprises one or more of HSPC, cholesterol, DSPC, sphingomyelin, DHSM, cholesterol and methoxy DSPE-PEG (preferably DSPE-PEG2000).

[0073] In one embodiment the lipid membrane comprises about 20-60 mol% HSPC or DSPC and / or about 5-60 mol% sphingomyelin or DHSM and / or about 20-60 mol% cholesterol and / or about 1-15 mol% DSPE-PEG.

[0074] In one embodiment the lipid membrane comprises 20-60 mol% HSPC or DSPC, about 20-60 mol% cholesterol, about 5-60 mol% sphingomyelin or DHSM and about 1-15 mol% DSPE-PEG.

[0075] In one embodiment, the DSPE-PEG is selected from DSPE-PEG2000 or DSPE-PEG5000. In one embodiment, the lipid membrane comprises HSPC or DHSM, cholesterol, and DSPE-PEG2000 in an about 56:39:5 mol% ratio.

[0076] In one embodiment, the lipid membrane comprises HSPC or DHSM, cholesterol, and DSPE-PEG2000 in an about 57:38:5 mol% ratio.

[0077] In one embodiment the lipid membrane comprises DSPC, sphingomyelin, cholesterol, and DSPE-PEG2000 in an about 100:5:5:4 mol ratio.

[0078] Liposomes are characterised by the amount (in moles or weight) of each component relative to the amount of lipid present in the lipid bilayer(s). For the avoidance of doubt, the total lipid present in the lipid bilayer includes sterols and lipopolymers as well as glycerophospholipids, sphingomyelins and other phospholipids. The mass of total lipid present in the liposomes can be calculated based on the amount of lipid used to prepare the liposomes. Where the lipid membrane comprises mostly phospholipids, the total lipid concentration can also be estimated using the Stewart assay (Stewart, 1980), which measures the phospholipid concentration. Where the lipid membrane comprises a significant proportion non-phospholipid compounds, such as sterols (e.g. cholesterol), the total lipid concentration in solution can be estimated by determining the phospholipid concentration using the Stewart Assay and dividing the determined concentration of phospholipid by the initial fraction of phospholipid :total lipid used in the preparation. Alternatively, the total lipid concentration can be determined by high performance liquid chromatography, quantitative mass spectrometry, nuclear magnetic resonance spectroscopy, or other such methods of quantification known to those skilled in the art. Once known, the total lipid concentration can be converted to mass and moles, using standard calculations.

[0079] In the method of the invention, it is advantageous for the liposomal apomorphine to have a high drug to lipid ratio, so that the same therapeutic benefit can be achieved using a lower dose.

[0080] In one embodiment the ratio of apomorphine to lipid is greater than 0.2:1 wt / wt.

[0081] In one embodiment the liposomal apomorphine has a drug to lipid ratio of between about 0.05: 1 to about 5:1 (mol / mol) and / or about 0.05: 1 to about 5:1 (wt / wt) apomorphine:total lipid constituents.

[0082] In one embodiment the ratio of apomorphine to lipid in the liposomal apomorphine is between about 0.05:1 and about 5:1 (mol / mol), about 0.1:1 to about 4:1, about 0.2:1 to about 3: 1 or about 0.4: 1 to about 2:1 (mol / mol).

[0083] In one embodiment the ratio of apomorphine to lipid is greater than 0.5:1 (mol / mol). In one embodiment the ratio of apomorphine to lipid is about 0.05: 1 to about 5:1 (wt / wt), about 0.1:1 to about 3:1, about 0.2:1 to about 2:1 or about 0.5:1 to about 1:1 (wt / wt).

[0084] High apomorphine to lipid ratios can be achieved by preparing liposomes comprising an ammonium salt and then using an ion gradient to replace the ammonium salt with apomorphine.

[0085] In one embodiment the liposomal apomorphine for use in the method of the invention is prepared by a process comprising:

[0086] (a) preparing an aqueous suspension comprising liposomes of 50-400 nm size wherein the liposomes comprise a lipid membrane enclosing an aqueous intraliposomal compartment containing an ammonium salt that is (i) at a concentration of about 150 mM to about 550 mM and (ii) that is greater than the concentration of ammonium salt in the aqueous suspension; (b) mixing the aqueous suspension with a solution of apomorphine salt wherein the apomorphine enters the intraliposomal compartment and forms a salt with anions of the ammonium salt, wherein the ratio of apomorphine in the intraliposomal compartment to the total lipid in the lipid membrane is about 0.05: 1 to about 5:1 (mol / mol) and / or about 0.05: 1 to about 5:1 (wt / wt);

[0087] (c) optionally removing unencapsulated apomorphine from the aqueous suspension. In the above method, in step (a) an aqueous suspension of liposomes is prepared. The liposomes are about 50-400 nm size and comprise a lipid membrane enclosing an aqueous intraliposomal compartment, which in turn comprises ammonium salt.

[0088] In one embodiment the concentration of ammonium salt inside the intraliposomal compartment is about 200 mM to about 500 mM. It is assumed that the concentration of ammonium salt inside the intraliposomal compartment is about the same as the concentration of ammonium salt used in the preparation of the liposomes.

[0089] In one embodiment the concentration of ammonium salt inside the intraliposomal compartment is about 200 mM to about 500 mM. In one embodiment the concentration of ammonium salt inside the intraliposomal compartment is about 250 mM to about 400, preferably about 150 to about 350 mM, more preferably about 250 mM to about 350 mM, most preferably about 300 mM.

[0090] In one embodiment the ammonium salt comprises ammonium and a multivalent counterion. In one embodiment the ammonium salt is selected from the group comprising ammonium sulfate, ammonium citrate, ammonium oxalate, ammonium phosphate, ammonium trimesate, sucrose ammonium octasulfate and

[0091] carboxyfl uoresecein ammonium salt.

[0092] The concentration of ammonium salt inside the intraliposomal compartment is also greater than the concentration of ammonium salt in the aqueous suspension. Methods of preparing liposomes of high ammonium salt concentration are known in the art, including in US Patents 5,316,771 and 11,413,244. An exemplary method is described in Example 1.

[0093] In one embodiment the aqueous suspension contains substantially no, or trace amounts of ammonium ions.

[0094] In one embodiment the aqueous suspension comprises an aqueous buffer. The aqueous suspension may also include one or more of sodium chloride, sucrose, antioxidants and preserving agents. In step (b) the aqueous suspension is mixed with a solution of apomorphine salt wherein the apomorphine is taken into the liposomes and forms a salt with anions of the ammonium salt.

[0095] Apomorphine salts are soluble in aqueous solution and dissociate to provide positively charged apomorphine ions at low pH. Where the pH is higher, apomorphine is largely deprotonated. Apomorphine has a pKa of 7.2.

[0096] Without being bound by theory, it is assumed that loading occurs as a complex equilibrium process between the dissolved ammonium salt and ammonia within the liposome, and protonated and unprotonated apomorphine external to the liposome, where the uncharged species can freely cross the liposome membrane resulting in the intraliposomal formation of insoluble apomorphine salt.

[0097] In one embodiment the apomorphine salt is selected from hydrochloride salt and methanesulfonate salt. Hydrochloride salt is preferred.

[0098] In one embodiment the solution comprises apomorphine salt in buffer. In one embodiment the solution comprises apomorphine salt in PBS buffer.

[0099] The concentration of apomorphine in the loading solution should be as high as practicable, taking into account the solubility of the apomorphine salt. In one embodiment the loading solution comprises about 4 mM apomorphine hydrochloride. In one embodiment the concentration of lipid in the aqueous liposome suspension is about 1 mg / mL to about 100 mg / mL, about 5 mg / mL to about 50 mg / mL, about 10 mg / mL to about 30 mg / mL. In another embodiment the concentration of lipid in the aqueous liposome suspension is about 10 mg / mL.

[0100] In one embodiment the aqueous suspension of liposomes is mixed with the solution of apomorphine salt in a ratio of about 1:100 to about 100:1 v / v, preferably about 1:20 to about 20:1. In one embodiment the apomorphine salt solution is about 4 mM concentration. Different ratios of liposomes to apomorphine salt give different effects. Assuming a liposome concentration of about 10 mg / mL, if the liposomal suspension is in excess, the system is driven to load all of the apomorphine into the liposomes. This leaves less apomorphine to be removed from the suspension but results in less apomorphine sulfate per liposome (and so a lower drug to lipid ratio). If the apomorphine is in excess, the system is driven to exhaust the ammonium sulfate gradient resulting in a higher concentration of apomorphine sulfate in the intraliposomal compartment (about equivalent to the concentration of ammonium sulfate in the liposomes). This results in a higher drug to lipid ratio but leaves excess apomorphine to be removed from the aqueous suspension. In one embodiment the liposomal suspension is mixed with the solution of apomorphine salt in a ratio of about 1:5 v / v.

[0101] The process of the invention utilises an ion gradient across the liposomal membrane to move apomorphine into the intraliposomal compartment, achieving a concentration of about 200 mM to about 500 mM of apomorphine salt in the intraliposomal compartment. As the concentration of ammonium salt increases, osmotic stress across the liposome membrane becomes a barrier to efficient drug loading and the preparation of a stable product. Consequently, it is preferred that the ammonium salt concentration in the liposomes not exceed about 550 mM.

[0102] This increased osmotic pressure exhibited across the liposome membrane can be balanced by the inclusion of appropriate salts, for example sodium chloride, or sugars, for example sucrose, in the loading solution containing the apomorphine salt or the aqueous suspension.

[0103] In one embodiment the solution of apomorphine salt comprises one or more of sodium chloride and sucrose. In one embodiment the concentration of sodium chloride or sucrose is approximately osmotically equivalent to the concentration of ammonium salt in the liposomes.

[0104] The pH of the mixture is important because it determines the ratio of protonated to deprotonated apomorphine. In one embodiment the pH of the mixture of the aqueous liposomal suspension and apomorphine salt solution is between about pH 5 and about pH 8. In another embodiment, the pH of the mixture is between about pH 6 and about pH 7. Where one of the two components is in excess, the pH of the mixture can be approximated by the pH of the excess component.

[0105] In one embodiment the aqueous suspension of liposomes comprising about 10 mg / L lipid, is mixed with the solution of apomorphine salt (preferably about 4 mM) in a ratio of about 1 : 100 to about 1 : 5 v / v and the pH of the solution of apomorphine salt is between about pH 5 and about pH 8. In another embodiment, the pH of the solution is between about pH 6 and about pH 7.

[0106] A person skilled in the art would know how long and under what conditions to mix the aqueous suspension of liposomes with the solution of apomorphine salt. For example, the opacity of the mixture can be monitored. As apomorphine salt precipitates in the intraliposomal compartment, the mixture becomes more opaque. The process is complete when the opacity stops changing.

[0107] In one embodiment the aqueous suspension of liposomes is mixed with the solution of apomorphine salt overnight at room temperature. This process results in liposomal apomorphine in which the ratio of apomorphine in the intraliposomal compartment to the total lipid in the lipid membrane of the liposome is about 0.05:1 to about 5:1 (mol / mol) and / or about 0.05:1 to about 5:1 (wt / wt).

[0108] Optionally in step (c) the unencapsulated apomorphine salt is removed from the aqueous suspension, for example, by dialysis or size exclusion chromatography.

[0109] The method of the invention relates to intranasal administration of an encapsulated apomorphine composition, in particular a liposomal apomorphine composition. In one embodiment the liposomal apomorphine composition comprises liposomal aposomes and one or more pharmaceutically acceptable excipients.

[0110] Pharmaceutically acceptable excipients are carriers that are safe, non-toxic and not biologically undesirable. In one embodiment the pharmaceutically acceptable excipient is an aqueous or non-aqueous, isotonic sterile solution, which may contain excipients such as anti-oxidants, buffering agents, and solutes including but not limited to saline, buffer, sugars, thickening agents, stabilisers and preservatives.

[0111] Suitable excipients include buffers, thickening agents and humectants. Examples of suitable buffers include but are not limited to acetate, citrate, prolamine, carbonate and phosphate buffers. Examples of suitable thickening agents include but are not limited to methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.

[0112] Examples of suitable humectants include but are not limited to sorbitol, glycerol, mineral oil, vegetable oil and combinations thereof.

[0113] The concentration of apomorphine in the liposomal apomorphine composition depends on its intended use but is sufficient to achieve a therapeutic effect upon administration to a subject in need thereof, wherein the amount of pharmaceutical composition administered is below the dose which would cause unacceptable side effects such as nausea, emesis, tachycardia and bladder pain. The amount of apomorphine required to achieve a therapeutic effect without causing unacceptable side effects depends on various parameters known to those skilled in the art, including the type and severity of disease, and the gender, age, weight and other determinants of the subject to be treated.

[0114] Achieving a therapeutic effect encompasses ameliorating undesired disease symptoms, preventing the manifestation of such symptoms before they occur, slowing down progression of the disease and / or symptoms, enhancing the onset of remission of the disease, slowing down damage caused by progressive chronic stages of the disease, delaying onset of progressive stages, lessening severity of the disease, improving the survival rate and / or curing or preventing the disease from occurring. In one embodiment the pharmaceutical composition is formulated as an intranasal dosage form. In one embodiment the dosage form comprises a total amount of apomorphine that is sufficient to provide a therapeutic effect upon nasal administration to a subject in need thereof. In one embodiment the subject is human. In one embodiment the therapeutic effect is provided within 60 minutes of administration. In one embodiment the condition is a neurological condition. In one embodiment the condition is Parkinson's disease. In one embodiment the condition is Restless Leg Syndrome. In one embodiment the condition is Erectile Dysfunction. In one embodiment the condition is Female Sexual Dysfunction. In one embodiment the condition is Alzheimer's disease and / or dementia.

[0115] In one embodiment the method provides a therapeutic improvement in motor function in the subject lasting at least 2-hours in duration.

[0116] 6. EXAMPLES

[0117] General Experimental Methods

[0118] Apomorphine fluorescence standard curve

[0119] 10 pM and 100 pM apomorphine solutions were prepared containing phosphate buffered saline and 2 mg mL-1ascorbic acid, and serially diluted to give final apomorphine concentrations between 0 and 9 pM, and 10 and 90 pM respectively. Fluorescence intensity from 100 pM aliquots of the solution were subsequently recorded on a CLARIOstar® fluorescence plate reader with an excitation and emission wavelength of 320 nm and 435 nm respectively using Nunc™ 96-well microtitre fluorescence plates. Additionally, the effect of liposomes and lysing surfactant myristryltrimethylammonium bromide (MTAB) was investigated through the addition of 10 pM of a 10 mM liposome suspension (aforementioned lipid formulation), or the addition of 10 pM of a liposome suspension with an additional 10 pM of 25 mM MTAB.

[0120] General procedure for TEM and Cryo-TEM experiments

[0121] Negative stain TEM was performed using a Philips CM100 BioTWIN transmission electron microscope (Philips / FEI Corporation, Eindhoven, Neatherlands) combined with a LakB 6 emitter, fitted with a MegaView III Olympus digital camera. Samples were prepared by depositing 10 pM of a diluted liposome suspension onto a 3 mm plasma-ionised carbon-coated copper specimen grid and removing it by capillary wicking using Whatman® filter paper after 60 seconds. Subsequently 10 pL of 1% phosphotungsted acid solution was deposited onto the grid and immediately removed by capillary wicking, and the grid was dried under heat lamp. Cryo-TEM was performed using a JEOL 2200FS field emission scanning electron microscope (STEM) with an omega energy filter (JEOL Ltd., Tokyo Japan), fitted with a TVIPS F416 CMOS camera (TVIPS GmbH, Germany) and Direct Electron DE-20 detector (Direct Electron LP, California, USA). Specimen holders used included a standard JEOL holder with a single tilt axis (JEOL Ltd., Tokyo, Japan) and a Gatan model 914 high tilt cryo holder (Gatan Inc., California, USA). Images were acquired using SerielEM software (University of Colorado, Colorado, Boulder, USA) in conjunction with TVIPS EM-Menu and DE Imaging Manager. Image analysis was performed using the IMOD software package (Boulder Laboratory for 3D Microscopy, Colorado, USA).

[0122] Example 1: Preparation and characterisation of liposomal apomorphine Liposomes were prepared using a variation of the thin-film rehydration method and adapted for active drug loading. Liposomes were generally prepared using a lipid composition comprising hydrogenated soy phosphatidylcholine, cholesterol and DSPE-PEG2000 in a 57:38:5 mol% ratio, or DSPC, sphingomyelin, cholesterol, DSPE-PEG2000 in a 100:5:5:4 mol ratio by combining the appropriate volumes of the membrane components dissolved in chlorinated solvent (e.g. chloroform, dichloromethane) (see Table 1) and subsequently removing the solvent in vacuo to form a lipid film.

[0123] Table 1: Lipid concentrations and volumes generally used in preparation of liposomes

[0124] Lipid Cone (mg mL1) Vol (M-L)

[0125] HSPC 16 987

[0126] Cholesterol 2 192

[0127] DSPE-PEG2000 10 224

[0128] DSPC 16 987

[0129] Sphingomyelin 4 200

[0130]

[0131] The dry lipid film was rehydrated with a solution containing the desired internal ammonium salt gradient, (for example: ammonium sulfate, ammonium citrate, ammonium oxalate, ammonium trimesate, carboxyfluorescein ammonium salt), a concentration of approximately 150 - 350 mM, without any additional buffering agents. To ensure dispersion of the lipid film, the suspension was heated to approximately the phase transition temperature of the lipid bilayer (approximately 50 °C) and vortexed or sonicated for five minutes, then extruded 15 times through polycarbonate membranes (1000 nm, 400 nm, 200 nm, and / or 100 nm in diameter) at 60°C to give a set of liposomes varying in size, ammonium salt concentration and ammonium salt anion (Olson, F., et al., 1979). The resulting liposomal suspension contained the same concentration of ammonium salt within and outside of the liposome. The external ammonium salt was removed via dialysis against phosphate buffered saline at pH 7.4. Dialysis was generally performed with 3 x 500 mL buffer changes at room temperature over 12 hours or until a sufficient ammonium salt gradient was established. The suspension of ammonium-containing liposomes in PBS was then stored at 4°C for later loading with apomorphine. The physical size characteristics of resulting ammonium sulfate liposomes are described in Table 2.

[0132] Table 2: Physical characterization of ammonium sulfate liposome preparation Liposome Batch Hydrodynamic Error Polydispersity Error Description Diameter (Z-Ave, nm) Index

[0133] 300 mM Ammonium 150 2 0.11 0.01 Sulfate

[0134]

[0135] The active loading process was initially investigated with 200 nm liposomes containing 300 mM ammonium sulfate (prepared as set out above). A 4 mM solution of apomorphine hydrochloride was prepared containing phosphate buffered saline (PBS), 2 mg mL-1ascorbic acid as an antioxidant at pH 6.3 and sodium chloride or sucrose in an approximately osmotically equivalent amount to the ammonium sulfate concentration in the liposomes. Apomorphine has a p / of 7.2, so loading was performed at pH 6.3 to ensure an adequate proportion of neutral apomorphine.

[0136] A 1 in 5 dilution of the liposome suspension (10 mg / mL lipid) was made using the apomorphine solution (400 p.L liposome suspension to 1.6 mL apomorphine solution in a 2 mL glass HPLC tube). Apomorphine loading was performed at room temperature over 12 hours in a sealed tube with stirring. Any residual unencapsulated apomorphine was subsequently removed by dialysis against PBS at pH 7.4 containing ascorbic acid (2 mg mL'1).

[0137] After the removal of any residual unencapsulated apomorphine by dialysis the baseline fluorescence of the liposomal apomorphine was measured using excitation and emission wavelengths of 320 and 435 nm respectively with and without the inclusion of myristrymethylammonium bromide (MTAB) to ascertain the approximate amount of apomorphine encapsulated. Over the 12 hour period, the liposomal apomorphine suspension was observed to turn noticeably opaque. Cryo-TEM was used to assess the resulting morphology of the apomorphine encapsulated liposome, as shown in Figure 1.

[0138] Example 2: Effect of blood plasma on the stability of liposomal apomorphine The purpose of this study was to investigate the impact of blood plasma on the stability of liposome encapsulated apomorphine. For in vivo and clinical applications, liposomes should ideally provide stable encapsulation of apomorphine in the presence of blood plasma with a minimal rate of leakage.

[0139] Liposomal apomorphine was prepared via active loading against a 300 mM ammonium sulfate gradient, or a 250 mM ammonium oxalate gradient as described in Example 1. A suspension of liposomal apomorphine sulfate or liposomal apomorphine oxalate was diluted 1 in 5 in either phosphate buffered saline, or clarified whole blood plasma obtained from sheep (30% blood plasma, 70% phosphate buffered [pH 7.4, 20 mM trisodium phosphate]), and the release of apomorphine was measured by fluorescence intensity as previously described, using myristrymethylammonium bromide (MTAB) to lyse the liposomes and obtain 100% release after 30 minutes of incubation.

[0140] It was surprisingly observed that approximately 80-100% of the encapsulated apomorphine was released from both liposomal apomorphine sulfate and liposomal apomorphine oxalate suspensions when incubated with 30% blood plasma. This was significantly higher than observed when liposomal apomorphine oxalate was incubated in phosphate buffered saline in the absence of sheep blood plasma. These results demonstrate significant instability of liposomal apomorphine when manufactured using either ammonium sulfate or ammonium oxalate. The results are displayed in Figure 2.

[0141] Example 3: Instability of liposomal apomorphine in blood plasma and mechanism of release

[0142] To further investigate the instability of liposomal apomorphine and the mechanism of apomorphine release in response to sheep blood plasma, the inventors manufactured liposomes co-encapsulating apomorphine and calcein. Calcein is a self-quenching fluorescent dye with excitation and emission wavelengths of 495 nm and 515 nm respectively. The excitation and emission wavelengths of calcein are outside that of apomorphine, allowing them to be monitored concurrently and independently of each other.

[0143] Preparation of ammonium calcein

[0144] Calcein was recovered from an aqueous solution of sodium calcein (500 mM) by acid precipitation with hydrochloric acid. The resultant calcein was filtered, dried, and subsequently and dissolved in ammonia solution to give a final concentration of 300 mM (pH 7.4). Preparation of liposomes co-encapsulating apomorphine and calcein Ammonium calcein is prepared via dissolving calcein (free acid) in aqueous solution and adjusted to pH 7 with the addition of ammonia solution. Ammonium calcein liposomes (300 mM) were prepared using the protocol described in Example 1.

[0145] Ammonium calcein liposomes were incubated with apomorphine (4 mM). The liposomes were extruded through 200 nm polycarbonate membranes and the excess ammonium calcein removed via dialysis against phosphate buffered saline.

[0146] The apomorphine-calcein liposomes became visibly opaque and transmission electron microscopy confirmed the presence of solid apomorphine-calcein co-precipitates.

[0147] Stability of apomorphine-calcein liposomes in sheep blood plasma

[0148] A suspension of apomorphine-calcein liposomes was diluted 1 in 20 in either phosphate buffered saline or clarified whole blood plasma obtained from sheep (30% blood plasma, 70% phosphate buffered [pH 7.4, 20 mM trisodium phosphate]), and the release of apomorphine and calcein was measured by fluorescence intensity as previously described. Fluorescence intensity was measured at 2 minutes, 15 minutes and 30 minutes, with Triton-XlOO used to lyse the liposome suspension and obtain 100% release. The results are presented in Figure 3.

[0149] The investigation confirmed that >90% of the encapsulated apomorphine was released from the liposome suspension when incubated with 30% blood plasma for 30 minutes. In comparison, only ~3% of the encapsulated apomorphine was released when incubated in phosphate buffered saline over the same time period. Moreover, the release of apomorphine occurs surprisingly fast, with >80% the encapsulated apomorphine released within the first 2 minutes. In contrast, the inventors further discovered that, while encapsulated apomorphine spontaneously releases from the liposome suspension, encapsulated calcein remains stably encapsulated over the 30-minute time period with little difference in release when incubated with phosphate buffered saline (2%) and blood plasma (3%). The fact that the calcein remains entrapped within the liposome indicates that the mechanism of release of apomorphine from the liposome does not involve destruction of the liposome construct. The apomorphine is released independently, leaving the calcein trapped.

[0150] The study was performed by the inventors using several apomorphine-calcein liposomes comprising alternative lipid compositions including HSPC: Cholesterol: DSPE-PEG2000 in a 57:38:5 mol% ratio, and DSPC: Cholesterol: DSPE-PEG2000 in a 55:40:5 mol% ratio. These demonstrated similar levels of apomorphine release. These results demonstrate significant inability of liposomes to retain encapsulated apomorphine in the presence of blood plasma, posing a significant challenge to the development of a liposomal apomorphine product for therapeutic use.

[0151] Example 4: Intranasal administration of liposomal apomorphine - Pilot Study Given the unexpected instability of liposomal apomorphine to blood plasma thereby conferring no benefit to the pharmacokinetics and pharmacodynamic properties of apomorphine, the inventor contemplated the use of liposomal apomorphine intranasally as an alternative route of administration.

[0152] A study was undertaken to test the efficacy of nasally administered liposomal apomorphine in the treatment of Parkinson's disease symptoms induced by MPTP administration in mice. Apomorphine hydrochloride was used as a control.

[0153] Twenty-four (24) male C57BI / 6 mice had Parkinson's disease symptoms induced by administration of MPTP on Day 0, as described below. Saline, liposomal apomorphine, or aqueous apomorphine were administered intranasally (IN), QD on Day 7 (T=0), as detailed in Table 3. On Day 7, mice were tested for motor coordination using the rotarod evaluation on Day 7 with all animals subsequently sacrificed via CO2 overdose and no terminal collections performed.

[0154] Materials and Methods:

[0155] Animals

[0156] Male C57BI / 6 mice (n=24; 6-8 weeks) with an average starting body weight (± SEM) of 23.99±0.39 g were obtained from Charles River Laboratories (Wilmington, MA, USA). Animals were acclimatized for a minimum of three days prior to study commencement. During this period, the animals were observed daily in order to reject any that presented in poor condition.

[0157] Housing

[0158] The study was performed in animal rooms provided with HEPA filtered air at a temperature of 21±4°C and 50%±20% relative humidity. Animals were housed in groups of up to 8 per cage. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour. The room was on an automatic timer for a light / dark cycle of 12 hours on and 12 hours off with no twilight. Bed-O-Cobs® or equivalent bedding was used. Cages, tops, and water bottles were washed with a commercial detergent and allowed to air dry. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number, and treatment group was used to mark all cages. The temperature and relative humidity were recorded during the study and the records retained.

[0159] Diet

[0160] Animals were fed with LabDiet 5053 rodent diet and water was provided ad libitum. Animal Randomization and Allocations

[0161] At the start of the study animals were randomized into three (3) groups of eight (8) animals each. Each animal was identified by an ear punch corresponding to an individual number. A cage card was used to identify each cage and was marked with the study number (MSY-01), treatment group number, and animal numbers.

[0162] Experimental Design

[0163] Prior to study start, liposomal apomorphine was prepared from liposomes containing 250 mM ammonium sulfate, as described in Example 1.

[0164] For the in-life component, twenty-four (24) male C57BI / 6 mice were used in. Parkinson's Disease symptoms were induced by administration of 18 mg / kg MPTP via intraperitoneal (IP) injections once every two (2) hours for a total of four (4) injections on Day 0.

[0165] Additional precautions were taken when handling MPTP and with the mice for up to five (5) days following administration.

[0166] Test article or vehicle were administered intranasally (IN), QD on Day 7 (T=0), as detailed in Table 3. On Day 7, mice were tested for motor coordination using the rotarod test 30 minutes, 2 hours, and 4 hours post-IN dose.

[0167] After the 4-hour rotarod evaluation on Day 7, all animals were sacrificed via CO2 overdose and no terminal collections were performed.

[0168] The details of the study design are shown in Table 3. Table 3: Study Design

[0169] MPTP

[0170] Group N° (Day Treatment Dose Schedule Route Rota rod O)

[0171] 1 8 Vehicle -- Day 18

[0172] mg / kg 7

[0173] Liposomal

[0174] 2 8 0.05 mL QD

[0175] IP Apomorphine

[0176] Day 7 IN

[0177] Every 2 30 minutes & hours T=0 2 hours post- Apomorphine

[0178] 3 8 0.05 mL IN 4 / day Hydrochloride administratio n

[0179]

[0180] MPTP preparation and disease induction

[0181] l-Methyl-4phenyl-l,2,3,6-tetrahydropyridine hydrochloride (MPTP; Sigma-Aldrich) was prepared. Doses were calculated as the free base, not the salt, form. MPTP was prepared using sterile saline (0.9%) and all injections were administered via the intraperitoneal cavity at a volume of 0.1 mL / 10 grams of body weight. All MPTP administrations were performed under a fume hood. Disposable cages were changed 3 days following MPTP administration in order to capture all MPTP and its active metabolite MPP+ which were excreted from the body during this time. Additional personal protective equipment (PPE) was worn during the high-risk period (administration and 5 days following). Required PPE included a disposable one-piece garment with hood, elastic wrists, and boots, respirator, gloves, and goggles. This PPE was worn anytime the technician was handling the mice, bedding, cages, or other materials the mouse or MPTP solution had come into contact with. All these materials were treated as biohazardous waste and were decontaminated with a 10% bleach solution prior to transport. Following all procedures, reusable equipment (weigh boats, necropsy tools, etc.) and surfaces (lab bench, cage rack, etc.) were decontaminated with a 10% bleach solution while PPE is worn.

[0182] Test-Articles and Formulations

[0183] Test Article Name Apomorphine Hydrochloride Physical Description Powder

[0184] Source: Tocris (Product #2073)

[0185] Vehicle: PBS (pH 6.3)

[0186] Route of Administration Intranasally (IN)

[0187] Dosing Details: Dose 0.1 mg / kg, 0.05 mL; QD, Day 7

[0188]

[0189] Test Article Name Liposomal Apomorphine

[0190] Physical Description Aqueous Solution

[0191] Vehicle: PBS (pH 6.3)

[0192] Route of Administration Intranasally (IN)

[0193] Dosing Details: Dose 0.1 mg / kg, 0.05 mL; QD, Day 7

[0194]

[0195] Note: Pegylated remote loadable liposomes contain 250 mM ammonium sulfate (internal liposome concentration), 25 mg / mL total lipid concentration, and have a size distribution of approximately 90 nm. The buffer composition contains 10 mM histidine buffer, pH ~6.5 with 10% w / v sucrose.

[0196] Body Weight and Survival

[0197] Animals were observed daily (weight, morbidity, survival) in order to assess possible differences among treatment groups and / or possible toxicity resulting from the treatments.

[0198] Rotarod

[0199] Mice were tested for motor coordination using a rotarod apparatus (Accuscan, Columbus, OH, USA). Mice were placed on a stationary dowel which accelerated at 1 rpm / 3 seconds until the mouse fell off or 3 minutes elapsed. The time until the mouse fell was automatically recorded by photobeams at the base of the rotarod. If the mouse did not fall within three (3) minutes, it was removed from the dowel and the time was recorded as 180 seconds. This procedure was repeated for four (4) consecutive trials with a minimum 30 second inter-trial interval.

[0200] Supportive care and euthanasia criteria

[0201] Animals in excess of 15% weight loss were administered 1 mL saline daily. Any animal that lost >20% of its body weight, showed an inability to eat, or was moribund, was euthanized. Animals that were found dead or were euthanized prior to scheduled termination days did not undergo terminal collections.

[0202] Sacrifice and Sample Collection

[0203] After the 4-hour rotarod evaluation on Day 7, all animals were sacrificed via CO2 overdose and no terminal collections were performed.

[0204] Survival

[0205] Animal deaths were evaluated during the course of the study. In this study, three (3) animals were found dead.

[0206] Outcome Evaluation

[0207] Study endpoints were body weight change and rotarod latency to fall. Statistical Analyses

[0208] Data were evaluated using one-way ANOVA with Dunnett's multiple comparisons test to compare all groups to the vehicle control group. All statistical analyses were performed using GraphPad Prism 9.3.1 (La Jolla, CA). Statistical significance was achieved when p<0.05.

[0209] Results and Discussion

[0210] Initial results suggest that liposome encapsulation can extend the mean latency-to-fall compared with apomorphine hydrochloride solution at an equivalent single-dose concentration (Figure 4).

[0211] This suggests an extension of dopaminergic agonist activity and therapeutic effect duration. The difference between groups can be observed after 30 minutes and 2 hours, which is in contrast to the rapid apomorphine release from liposomes when incubated with blood plasma observed in Experiment 3. Therefore, liposomal apomorphine provides a significant improvement over the standard of care when administered intranasally, reaching a therapeutic duration equivalent to or greater than that of L-DOPA (~90 minutes), the current standard of care. 7. REFERENCES

[0212] Dewey, R., Maraganore, D., Ahlskog, J., & Matsumoto, J. (1996). Intranasal apomorphine rescue therapy for parkinsonian "off" periods. Clin Neuropharmacol, 19(3), 193-201. doi:https: / / doi.org / 10.1097 / 00002826-199619030-00001 Dewey, R., Maraganore, D., Ahlskog, J., & Matsumoto, J. (1998). A double-blind, placebo-controlled study of intranasal apomorphine spray as a rescue agent for off-states in Parkinson's disease. Mov Disord, 13(5), 782-787.

[0213] doi: https: / / doi.org / 10.1002 / mds.870130505

[0214] Kapoor, R. T. (1990). Intranasal apomorphine: a new treatment in Parkinson's disease. J Neurol Neurosurg Psychiatry, 1015, 1015. doi:https: / / doi.org / 10.1136 / jnnp.53. Lasic, M. C. (1992). Biochim. Biohys.Acta,, 113, 171-199.

[0215] Levin, Y., & Idiart, M. A. (2004). Pore dynamics of osmotically stressed vesicles. Physica A: Statistical Mechanics and its Applications, 331 (3-4), 571-578.

[0216] Navarro, C. S. (2011). Liposomes for Targeted Delivery of Active Agents against Neurodegenerative Diseases (Alzheimer's Disease and Parkinson's Disease). J Drug Deliv. doi: 10.1155 / 2011 / 469679

[0217] O, G. (2005). Chem Phys Lipids, 135, 117-129.

[0218] Olson, F., et al. (1979). Biochimica et Biophysica Acta (BBA) - Biomembranes, 557 (1). Priev, A., et al. (2002). Langmuir 18, 612-917.

[0219] Shmeeda, H., Even-Chen, S., Honen, R., Cohen, R., 8<. Weintrau. (2003). Enzymatic assays for quality control and pharmacokinetics of liposome formulations: comparison with nonenzymatic conventional methodologies. Methods Enzymol, 367, 272-92.

[0220] Stewart. (1980). Retrieved from https: / / pubmed.ncbi.nlm.nih.gov / 6892980 /

Claims

WHAT WE CLAIM:

1. A method of treating a neurological disorder in which an increase in dopaminergic turnover is beneficial, the method comprising intranasal administration of an encapsulated apomorphine composition to a subject in need thereof.

2. A method of claim 1 in which the encapsulated apomorphine composition comprises one or more encapsulated apomorphine constructs selected from the group comprising liposomal apomorphine, polymeric apomorphine nanoparticles, lipid apomorphine nanoparticles, protein-encapsulated apomorphine, protein-bound apomorphine, and mesoporous nanoparticles of apomorphine.

3. A method of claim 2 in which the encapsulated apomorphine composition comprises liposomal apomorphine.

4. A method of claim 3 in which the ratio of apomorphine in the intraliposomal compartment to the total lipid in the lipid membrane is about 0.05: 1 to about 5:1 (mol / mol) and / or about 0.05: 1 to about 5:1 (wt / wt).

5. A method of any preceding claim wherein the disorder is Parkinson's disease.

6. A method of claim 5 which provides a therapeutic improvement in motor function in the subject lasting at least 2-hours in duration.

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