The invention relates to liposomal-encapsulated formulations of a compound of formula I. The invention further relates to a pharmaceutical composition thereof and its use in the treatment of neurodegenerative diseases and related conditions.

BACKGROUND ART

Neurodegenerative diseases are incurable and debilitating conditions that result in progressive degeneration and/or death of nerve cells, primarily affecting the neurons. This causes problems with movement (ataxias) and/or mental functioning (dementias). Examples of neurodegenerative diseases include Alzheimer's (AD), Parkinson's (PD), Huntington's (HD), Lou Gehrig’s (Amiotrophic Lateral Sclerosis -ALS) and other neurodegenerative conditions resulting in Dementia, Movement Disorders and other symptoms and disabilities. Similarly, there exist certain inherited conditions where cognitive impairment and progressive dementia associated to neurodegeneration might occur. 21 trisomy (Down syndrome) and X-fragile syndrome are the two most frequent conditions leading to cognitive disability, however, other similar conditions also exist.

Neurodegenerative diseases are generally considered to be irreversible at the present time. To date, no curative treatments exist. Present treatments, to the extent they exist, focus on symptom maintenance, or palliative care, rather than on a cure. As described herein, existing products for such palliative care have a number of drawbacks.

Currently available treatments offer relatively limited, symptomatic benefit and remain palliative in nature although they may be of value to improve the patient’s quality of life. As these are chronic and progressive disorders, there exists a strong need for a treatment that slows or stops disease progression. Current treatments also have a limited time in which they are efficacious. As a result, there also exists a need for a treatment that has a more prolonged efficacy, as well as reduced side effects.

Levodopa therapy is currently the primary treatment for Parkinson's Disease. However, Levodopa therapy has existing drawbacks as described herein. Levodopa therapy causes a number of adverse effects, among which dyskinesia, which is characterized by involuntary muscle movement is one of the most inhabilitating for patients.

Alternative compositions aimed at ameliorating certain of the drawbacks discussed above have been described. For example, U.S. Patent Application Number 20170296491 A1 discloses a Levodopa amide compound used in the treatment of diseases characterized by neurodegeneration. That patent discloses a formulation with improved brain permeability, which potentially reduces the necessary dosage of Levodopa, and therefore reduces the side effects of the treatment. Similarly, U.S. Patent US6500867B1 discloses an oral solid fixed dose composition comprising entacapone, Levodopa, and carbidopa that may be used in treatment of neurodegenerative diseases. That patent discloses a single fixed dose medication, which eliminates the need for patients to take multiple tablets several times a day.

It is known in the art that inflammation is an important underlying contributor to neurodegenerative diseases. Similarly, oxidative processes are thought to play important roles in neurodegeneration. Therefore anti-inflammatory and antioxidant agents may serve as disease modifying treatments. Research has shown that compounds with anti-inflammatory and antioxidant capacity such as Oleoylethanolamide (OEA) might attenuate both pathogenic mechanisms (see Anton M, Rodriguez de Fonseca F et al. Oleoylethanolamide prevents neuroimmune HMGB1/TLR4/NF-kB danger signaling in rat frontal cortex and depressive-like behavior induced by ethanol binge administration. Addiction Biology. 2017 May; 22(3):724-741.). However, to date, there still does not exist an effective treatment that utilizes the beneficial properties of anti-inflammatory or antioxidant compounds to treat a broad spectrum of neurodegenerative diseases while minimizing side effects.

The endocannabinoid system has likewise been studied for years for its antiinflammatory and homeostatic properties. A structural analogue of the endocannabionoid anandamide belonging to the N-acylethanolamide family, the lipid mediator oleoylethanolamide (OEA), has emerged as an interesting bioactive molecule with anti-inflammatory and neuroprotective actions in the brain. However, orally administered OEA suffers a substantial degradation in the gastrointestinal tract, and what survives presents a considerably low absorption rate in the intestine (Nielsen et al. Food intake is inhibited by oral oleoylethanolamide. June 2004, Journal of Lipid Research, 45, 1027-1029).

To surpass these limitations the need for an effective carrier or drug delivery system arises. To enable the development of potentially beneficial new drugs from lipophyllic molecules with useful physiological activities, a number of lipid-based drug carrier systems have been studied (see, for example, Kalepu, Sandeep & Manthina, Mohanvarma & Padavala, Veerabhadhraswamy. (2013). Oral lipid-based drug delivery systems - An overview. Acta Pharmaceutica Sinica B. 3. 10.1016).

Liposomes have been thought to have the capacity to increase the transport of active ingredients into the intestinal lymphatic circulation. However, liposomes have been reported inadequate for oral drug delivery in general, and it is thought that significant additions and modifications may be required to obtain effective oral liposomal formulations (see Wu et al., 2015 Ther. Delivery, 6 (11); He et al., 2018 Acta Pharm.Sin. B.,). In addition, it is known in the art that liposomes are easily disrupted by amphiphilic molecules such as OEA, and that the stability of liposomes formulated with OEA may be subject to , limitations on the maximum fractional amount of OEA that is encapsulated in the lipid phase relative to the lipids constituting the liposome bilayers. In actual practice obtaining effective liposomal formulations for drug delivery appears not to be straightforward, as liposomal delivery is available only for intravenous delivery of a select number of drugs in the market and no examples of any liposomal formulation for oral administration have been approved as drugs (see Bulbake, U., Doppalapudi, S., Kommineni, N., & Khan, W. (2017) Liposomal Formulations in Clinical Use: An Updated Review. Pharmaceutics, 9(2), 12).

In the particular case of OEA the priorities for an effective drug delivery formulation are namely to achieve i) formulation stability, ii) a significantly high degree of encapsulation of the active ingredient and iii) oral bioavailability allowing the active ingredient to reach and act on its physiological targets and mechanisms in animal models, in this case, OEA. A nano-formulation of OEA has been proposed trying to solve this approach (see Wullf-Perez et al. , Nanomedicine (Lond). 2014 Dec;9(18):2761-72), wherein OEA nanoemulsion were formulated which necessitated the use of synthetic surfactants. Moreover, the need for a large proportion of synthetic surfactants in such formulations indicates potential for serious adverse events in human subjects and dictates the need to explore its long term toxicity in animals before attempting to study it in humans. However this formulation is not stable for more than a few days, thus precluding further development as a potential drug or component of a pharmaceutical composition

Accordingly, there is a need for a treatment with more stable formulations for drug delivery that can be used to administer active NAEs, that can be maintained over time, without significant adverse effects, and that can allow effective delivery to the organism of sufficient N-acylethanolamides (NAEs), such as OEA, to effect its pharmacological activity in living animals, .

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to an aqueous suspension of liposomes for the administration of N-acylethanolamide (NAE) of formula I, wherein the liposomes present a multi-lamellar or unilamellar structure having a negative zeta potential between -10 mV and -100mV and maximum diameter of 300 nm; wherein the compound of formula I is encapsulated at a minimum rate of 80%; wherein the resulting concentration of the aqueous suspension has at least 60 mg, preferably of at least 300 mg, of a compound of formula I per 10 ml of the aqueous suspension of liposomes, and; wherein the resulting suspension comprises:

a) an aqueous phase;

b) an organic phase comprising the lipid components which form the multi-lamellar or unilamellar liposomes and accounts for 5% to 50% w/v of the total suspension; a natural antioxidant; a natural toughener; and a compound of formula I:

HI

wherein:

n is an integer ranging from 0 to 5;

a and b are determined by the following formula: 0 £ (a + b) £ 4;

Z is a group selected from -C(0)N(R ¹ )-, -(R ¹ )NC(0)-, -(O)CO-, O, NR ¹ and S;

R ¹ and R ² are independently selected from the group consisting of hydrogen, substituted or unsubstituted (CrC ₆ ) alkyl, substituted or unsubstituted (CrC ₆ ) acyl and aryl; and

wherein up to eight hydrogen atoms of the compound of formula I may be substituted by methyl or a double bond and the molecular bridge between c and d may be saturated or unsaturated,

or any of its pharmaceutically acceptable salts, esters, isomers, tautomers, stereoisomers, polymorphs, solvates, hydrates, derivatives, prodrugs or any combination thereof.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the compound of formula I is the compound of formula II:

wherein:

n is an integer ranging from 0 to 4;

a and b are determined by the following formula: 0 £ (a + b) £ 3;

R ¹ and R ² are independently selected from the group consisting of hydrogen, substituted or unsubstituted (CrC ₆ ) alkyl and substituted or unsubstituted (CrC ₆ ) acyl; and

wherein up to eight hydrogen atoms of the compound of formula II may be substituted by methyl or a double bond and the molecular bridge between c and d may be saturated or unsaturated,

or any of its pharmaceutically acceptable salts, esters, isomers, tautomers, stereoisomers, polymorphs, solvates, hydrates, derivatives, prodrugs or any combination thereof.

In another embodiment of the above mentioned aspect, the invention relates to the aqueous suspension as described above, wherein a = 1 and b = 1.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein n is 0 or 1 in formula I. In another embodiment, the invention relates to the aqueous suspension as described above, wherein R ¹ and R ² are hydrogen.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the bridge between the c carbon and the d carbon is a double bond.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the compound of formula I, preferably selected from oleoylethanolamide (OEA), palmitoylethanolamide (PEA), stearoylethanolamide (SEA), anandamide (AEA), linoleyl ethanolamide (LEA), and any combination thereof, and more preferably wherein the compound of formula I is oeloylethanolamide (OEA), or any of its pharmaceutically acceptable salts, esters, isomers, tautomers, stereoisomers, polymorphs, solvates, hydrates, derivatives, prodrugs or any combination thereof, is in an amount corresponding to between 2%(w/v) and 20%(w/v) of the total suspension.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the compound of formula I is selected from oleoylethanolamide (OEA), palmitoylethanolamide (PEA), stearoylethanolamide (SEA), anandamide (AEA), linoleyl ethanolamide (LEA), and any combination thereof.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the compound of formula I is oeloylethanolamide (OEA), or any of its pharmaceutically acceptable salts, esters, isomers, tautomers, stereoisomers, polymorphs, solvates, hydrates, derivatives, prodrugs or any combination thereof.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the compound of formula I is oleoylethanolamide (OEA) and is present in the suspension in a quantity from 4 to 36 % (w/w) of total lipids.

The preferred phospholipid materials for the invention are a combination of phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols and phosphatidic acids, to obtain maximum possible encapsulation of the active ingredient and also membrane strength. These materials are obtained from edible, nontoxic plant sources, including soy lecithin, sunflower lecithin, and their combinations.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the lipid components are selected from phosphatidylcholines (PCs), phosphatidylserines (PSs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), phosphatidylinositols (Pis), phosphatidic acids (PAs), and any combination thereof.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the lipid components are obtained from soy lecithin, sunflower lecithin, or any combination thereof, and preferably obtained from soy lecithin.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the lipid components further contain fatty acid and their triglycerides.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the soy lecithin is in a quantity from 8 g to 32 g per 100ml of suspension.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the natural antioxidant is selected from alpha-tocopherol (vitamin E), gamma-tocopherol, ascorbic acid, ascorbyl palmitate, gallic acid, protocatechuic acid, resveratrol, propyl gallate, epicatechin, epicatechin gallate, epigallocatechin gallate, rutin, crocin, safrole, anthocyanidin-3,5-glucoside, quercetin, and any combination thereof, and preferably wherein the natural antioxidant is alpha-tocopherol (vitamin E).

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the natural antioxidant is alpha-tocopherol (vitamin E) and is present in the suspension in a quantity from 1 to 30% of total lipids.

The preferred natural toughener for the invention is cholesterol, but other membrane active agents capable of modulating lipid bilayer properties such as fluidity and rigidity can be used, such as steroids, vitamins and other sterols derivatives such as phytosterols, stands and bile salts in appropriate ratios.

Accordingly, in another embodiment, the invention relates to the aqueous suspension as described above, wherein the natural toughener is selected from cholesterol steroids, vitamins, phytosterols, stands and bile salts, and preferably wherein the natural toughener is cholesterol.

In another embodiment, the invention relates to the aqueous suspension as described above, wherein the natural toughener is cholesterol and is present in the suspension in a quantity from 5 to 50% of total lipids.

Another aspect of the invention relates to the aqueous suspension as described above for use as medicament.

Another aspect of the invention relates to the aqueous suspension as described above for use in the treatment and/or prevention of diseases or conditions which are mediated by modulating neuro-inflammation.

In another embodiment of the above mentioned aspect the invention relates to the aqueous suspension as described above for use in the treatment and/or prevention of neurodegenerative diseases, more preferably for use in the treatment and/or prevention of neurodegenerative diseases selected from Alzheimer's, Parkinson's, Huntington's, Lou Gehrig's and Friedreich's diseases.

In another embodiment the invention relates to the aqueous suspension as described above for use in the treatment and/or prevention of inherited neurological conditions that generate progressive cognitive impairment throughout life, and preferably for use in the treatment and/or prevention of cognitive impairing inherited neurological conditions selected from Down syndrome and X-fragile syndrome.

Another aspect of the invention relates to the aqueous suspension as described above for the treatment and/or prevention of adverse effects derived from drugs and/or treatments currently used to treat diseases such Levodopa-induced dyskinesia suffered by patients having Parkinson's disease, and preferably wherein the compound of formula I of the aqueous suspension as defined above is oleoylethanolamide (OEA).

Another aspect of the present invention relates to a method of treating and/or prevention of diseases or conditions which are mediated by modulating neuro inflammation, in a subject in need thereof, especially a human being, which comprises administering to said subject an effective amount of the aqueous suspension as defined above or a pharmaceutically acceptable salt thereof. Another aspect of the present invention relates to a method of treating and/or prevention of neurodegenerative diseases, more preferably for use in the treatment and/or prevention of neurodegenerative diseases selected from Alzheimer's, Parkinson's, Huntington's, Lou Gehrig's and Friedreich's diseases, in a subject in need thereof, especially a human being, which comprises administering to said subject an effective amount of the aqueous suspension as defined above or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention relates to a method for the treatment and/or prevention of inherited neurological conditions that generate progressive cognitive impairment throughout life, and preferably for use in the treatment and/or prevention of cognitive impairing inherited neurological conditions selected from Down syndrome and X-fragile syndrome, in a subject in need thereof, especially a human being, which comprises administering to said subject an effective amount of the aqueous suspension as defined above or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention relates to a method for the treatment and/or prevention of adverse effects derived from drugs and/or treatments currently used to treat diseases such as Levodopa-induced dyskinesia suffered by patients having Parkinson's disease, and preferably wherein the compound of formula I of the aqueous suspension as defined above is oleoylethanolamide (OEA), in a subject in need thereof, especially a human being, which comprises administering to said subject an effective amount of the aqueous suspension as defined above or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to use of the aqueous suspension as described above for the creation and/or manufacture of a dietary supplement, medical food or nutraceutical.

Another aspect of the invention relates to a pharmaceutical composition which comprises the aqueous suspension as described above or any of its pharmaceutically acceptable salts, esters, isomers, tautomers, stereoisomers, polymorphs, solvates, hydrates, derivatives, prodrugs or any combination thereof; and one or more additional therapeutic agents, optionally together with one or more pharmaceutically acceptable carriers and/or diluents.

In another embodiment the invention relates to the pharmaceutical composition as defined above, administrated along with additional agents, and preferably wherein the additional therapeutic agents are selected from Levodopa, carbidopa and dopamine agonists.

Another aspect of the invention relates to the process to obtain the aqueous suspension as defined above which comprises the steps of:

a) preparing an aqueous phase comprising a buffer with a range of pH between 3.5 and 8.0 dissolved in distilled water and compatible with oral drug administration applications, preferably wherein the buffer is sodium citrate.

b) preparing separately an organic phase by dissolving in a water miscible solvent, preferably ethanol, the compound of formula I, in a concentration between 0.1% and 5.0% w/v, together with lipid components, a natural antioxidant and a natural toughener;

c) injecting said organic phase into said aqueous phase stirring constantly and maintaining the temperature between 50 °C and 65 °C and the pressure between 13.8 bar and 41.4 bar to encapsulate the active compound in first oligo-lamellar vesicles to obtain a liposomal dispersion; and

d) extruding the liposomal dispersion obtained in the previous step through membranes from 100nm to 300 nm pore size, preferably through membranes of 200 nm pore size.

In another embodiment the invention relates to the process to obtain the aqueous suspension as defined above, which further comprises the step of:

e) replacing the aqueous phase while reducing solvent concentration below 5%w/v by tangential ultra-filtration; and concentrating the solution obtained in the previous step to the final required concentration by additional tangential ultra-filtration. In another embodiment the invention relates to the process to obtain the aqueous suspension as defined above, which comprises the steps of:

a) preparing an aqueous phase comprising a buffer with a range of pH between 3.5 and 8.0 dissolved in distilled water and compatible with oral drug administration applications, preferably wherein the buffer is sodium citrate;

b) preparing separately an organic phase by dissolving in a water miscible solvent, preferably ethanol, the compound of formula I, in a concentration between 0.1% and 5.0% w/v, together with lipid components, a natural antioxidant and a natural toughener;

c) injecting said organic phase into said aqueous phase stirring constantly and maintaining the temperature between 50 °C and 65 °C and the pressure between 13.8 bar and 41.4 bar to encapsulate the active compound in first oligo-lamellar vesicles to obtain a liposomal dispersion;

d) extruding the liposomal dispersion obtained in the previous step through membranes from 100nm to 300 nm pore size, preferably through membranes of 200 nm pore size; and

e) replacing the aqueous phase while reducing solvent concentration below 5%w/v by tangential ultra-filtration; and concentrating the solution obtained in the previous step to the final required concentration by additional tangential ultra-filtration.

The aqueous suspension of the present invention can be administered in the form of any pharmaceutical formulation, the nature of which, as it is well known, will depend upon the nature of the aqueous suspension and its route of administration. The preferred route of administration of the aqueous suspension of the invention is oral administration.

Accordingly, in another embodiment the invention relates to the pharmaceutical composition as described above orally administrated.

Along the present invention the term“(CrC ₆ ) alkyl” as a group or part of a group, means a straight or branched alkyl chain which contains from 1 to 6 carbon atoms and includes, among others, the groups methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec- butyl, terf-butyl, pentyl and hexyl.“(CrC ₆ ) alkyl” may be optionally substituted by one or more, preferably by one or two substituents such as, among others, halogen, hydroxyl and (CrC ₆ ) alkoxyl.

The term“(CrC ₆ ) acyl” refers to a groups“(CrC ₆ ) alkyl-C=0”, wherein“(CrC ₆ ) alkyl” has the meaning stated above and wherein“(CrC ₆ ) acyl” may be optionally substituted as a“(CrC ₆ ) alkyl” as defined above. Examples include, among others,

The term “aryl” refest to monocyclic or bicyclic aromatic rings. Examples include, among others, phenyl, substituted phenyl and the like, as well as groups which are fused, such as, among others, napthyl, phenanthrenyl and the like. Aryl groups may optionally be substituted with one or more groups, preferably one or two, including, among others, halogen, (CrC ₆ ) alkyl, (CrC ₆ ) alkoxyl, hydroxyl, carboxyl, carbamoyl, alkoxycarbonyl, nitro, trifluoromethyl, amino, cyano or thiol.

The term “negative zeta potential” is measured in milivolts (mV) and refers to the degree of electrostatic repulsion between adjacent, similarly charged liposomes in dispersion. Values of negative zeta potential are between -1 mv and -100mV.

A natural toughener includes, among others cholesterol, steroids, vitamins, phytosterols, stands and bile salts.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration and are not intended to be limiting of the present invention. Examples

Example 1. Preparation of liposome- encapsulated OEA

Two solutions are prepared as the starting point for this elaboration:

Solution (A)

41 ,2 ml of an ethanol solution containing:

a. 184,3mg OEA

b. 72, 6mg of alpha-tocopherol

c. 941 , 7mg of cholesterol

d. 3.21 g of soy lecithin phospholipids

Solution (B)

408 ml of an aqueous saline solution citrate-buffered at pH 5.6 with total sodium at 0.15M

Solution (A) is injected into solution (B) while purging oxygen by using Nitrogen gas at 2 bar while stirring constantly with a 4 blade propeller on an IKA Eurostar 20 at 600 rpm and maintaining the temperature constant in a thermostatic bath at 60°C.

The dispersion thus obtained is extruded though polycarbonate membranes in 3 steps using progressively smaller pore size (from 5micron to 0.4 micron). A 100ml Lipex, extruder is used for this purpose pressurized by a N ₂ gas manostat controlled system at 27.6 bar., and maintained at 60°C by circulating thermostatic bath water through its base.

The extruded dispersion is subjected to tangential ultrafiltration through a 0.05micron mebrane against 20 volumes of aqueous pH 5.6 / citrate-buffered 0.9% saline solution a Pellicon countercurrent tangential filtration unit at 2 bar against the citrate buffered physiological saline solution (B). The solvent removal is followed by a concentration run to to achieve a suspension of approximately 15-25% of the original volume which is approximately 20% w/v in lipid components which can be performed on the same tangential filtration unit. Batches of this formulation are stored at 4°C. Example 2. Alternative liposome formulation A liposome preparation was performed by following the procedure of example 1 except for the composition of the starting solutions which was the following:

Solution (A)

41 ml of an ethanol solution containing

a. 711 ,5mg OEA

b. 82, 3mg of alpha-tocopherol

c. 623, 2mg of cholesterol

d. 3.11 g of soy lecithin phospholipids

Solution (B)

410 ml of an aqueous saline solution citrate-buffered at pH 5.6 with total sodium at 0.15M

This is processed by following essentially the same procedure as in example 1.

Example 3. Alternative lipid based formulation

As an alternative lipid drug delivery formulation, nanoemulsions were tested.

25% v/v sunflower oil-in-water nanoemulsions are prepared with 1 % (weight [w]/v) of surfactant poloxamer P188. Two batches are prepared sequentially by mixing together the solutions indicated in a high-speed stirrer (such as a Heidolph Diax 900) for 4 min at 13,000 rpm.

For the vehicle-only batch:

• 30 ml of sunflower seed oil, food grade.

• 10 ml 10% w/v of poloxamer P188 solution

• 68 ml of 0,2M sodium chloride/ 0,05M pH 5.6 sodium citrate buffer

• 2ml of absolute ethanol

For the OEA batch:

• 30 ml of sunflower seed oil, food grade.

• 10 ml 10% w/v of poloxamer P188 solution

• 58 ml of 0,2M sodium chloride/ 0,05M pH 5.6 sodium citrate buffer

• 2ml of 100mg/ml OEA in absolute ethanol These coarse emulsions are immediately homogenized using a high-pressure EmulsiFlex C3 homogenizer for ten passes at 100 MPa, and stored at 4°C.

Example 4. Characterization and stability of formulations

Particle size and monodispersity are determined by dynamic light scattering measurement. A high performance particle sizer, Turbiscan MA is used for the nanoemulsions by scanning (bottom to the top) with an 850 nm infrared light and backscattered light (45° from incident) recorded.

For liposome characterization a Zetasizer instrument is used for dynamic light scattering analysis. The electrophoretic cells of this instrument are used applying a 100mv potential for zeta potential determination.

Changes >20% in the measurements were taken as indication of loss of stability by coalescence, flocculation, or sedimentation.

The ratio of encapsulation or particle-association obtained is measured by first separating free from liposome- associated OEA by centrifugation at 20,000 g for 1 hour at 4°C in a refrigerated centrifuge. The pellets formed are washed twice with 10 mL bicarbonate buffer (pH 9.0) and recentrifuged again for 1 hour.

Total lipids are extracted from samples according to the Bligh and Dyer method with a mixture of chloroform-methanol (2:1 [v/v]) solution and butylated hydroxytoluene (0.025% [w/v]).

Extracts are subject to high-performance liquid chromatography/mass spectrometry (HPLC/MS).

Results

Liposomes formed by the procedures detailed in examples 1 and 2 have a negative zeta potential of -19 to -11 mV, and a mean diameter of 160-180nm as confirmed by electrophoretic dynamic light scattering analysis with a Zetasizer instrument. Such liposome preparations show prolonged stability when stored at 4°C for up to six months without significant changes. - While the freshly made nanoemulsions of example 3 presented a relatively homogeneous mean size in the 200nm range and were stable enough for immediate experimental use within 24-48 hrs, upon repeated attempts the size range distribution quickly became extremely broad (20nm- >1000nm without discernible peaks) even at 4°C. It was not possible to reproducibly achieve stability of nanoemulsions beyond 2-3 days at the most.

- The ratio of active agent entrapment/ encapsulation is found be above 95% of total OEA in all cases in which an average lipid vesicle size in the nanometer range could be maintained. When a significant fraction of the size distribution was below 100nm, the % encapsulation values decreased below 50%.

Example 5. Bioassav of OEA formulations

Wistar rats (groups of 5) are treated of formulation with OEA by intragastric gavage and compared to animals receiving vehicle (formulation components without active ingredients).

Rats are maintained with water but no food for 24 h. Formulated OEA- or vehicle-only formulation- (2ml) are then administered by intragastric gavage with a 5cm round tipped stainless steel needle 20 min before access to food, following accepted animal handling practices. Upon return to cages, a measured amount of food (50g) is delivered to the animals. Food is weighed at time of setting the animals in fresh cages and subsequently at intervals of 15, 30, 60, 120 and 240 min, and 24 h, and cumulative food intake is calculated as the difference between input and output.

Standard statistical analysis tools are used to detect significant differences and correlations in the data obtained.

Results

Analysis of the effect of OEA formulated according to examples 1 , 2 and 3 administered by gavage (1 , 10 and 50 mg/kg) and tested on feeding behavior at different times (30min, 60min, 2hrs, 6hrs, 24hrs), revealed a significant decrease (- 12%) of cumulative food intake initially detectable after the 60 min timepoint with 10 and 50mg/kg doses when compared with the vehicle group. Control of standard blood chemistry reveals no significant safety alerts within this time span, although the nanoemulsion of example 3 shows a small but significant increase in blood cholesterol (5% over saline alone).

Similarly, repeat of the testing the same formulations stored at 4°C for 30 days after they preparation shows statistically equivalent results for the formulation of example 1.