阅读说明：本技术 人工泪液 (Artificial tear ) 是由 L·菲茨亨利 S·库马里 于 2019-11-05 设计创作，主要内容包括：本发明涉及一种纳米结构脂质载体(NLC)颗粒以及相关组合物、滴眼液分配器、治疗或预防受试者干眼失调症的用途和方法,所述纳米结构脂质载体(NLC)颗粒包括：(i)包含固体脂质的固体外壳,其中所述固体脂质包含胆固醇；和(ii)包含液体脂质的液体核,其中所述液体脂质包含甘油三酯。(The present invention relates to a Nanostructured Lipid Carrier (NLC) particle comprising: (i) a solid shell comprising a solid lipid, wherein the solid lipid comprises cholesterol; and (ii) a liquid core comprising liquid lipids, wherein the liquid lipids comprise triglycerides.)

1. A Nanostructured Lipid Carrier (NLC) particle comprising:

(i) a solid shell comprising a solid lipid, wherein the solid lipid comprises cholesterol; and

(ii) a liquid core comprising liquid lipids, wherein the liquid lipids comprise triglycerides.

2. The NLC particle of claim 1, wherein the triglyceride comprises ricinoleic acid as the major fatty acid component.

3. The NLC particle of claim 1 or claim 2, wherein the triglyceride contains at least 85% ricinoleic acid.

4. The NLC particles of any preceding claim, wherein the triglycerides comprise ricinoleic acid, oleic acid, and linoleic acid.

5. The NLC particle of any one of the preceding claims, wherein the triglyceride is a castor oil triglyceride.

6. The NLC particles of claim 1, wherein the NLC particles are suitable for treating or preventing a dry eye disorder.

7. The NLC particles of any preceding claim, wherein the dry eye disorder comprises a condition selected from dry eye (keratoconjunctivitis sicca); conjunctivitis; keratitis; uveitis; scleritis; episcleritis; blepharitis; meibomian gland dysfunction; and iritis; or any combination thereof.

8. The NLC particle of any one of the preceding claims, wherein the Nanostructured Lipid Carrier (NLC) does not comprise or encapsulate a therapeutic agent.

9. The NLC particle of any preceding claim, wherein the NLC particle is nanoparticle sized.

10. The NLC particle of any preceding claim, wherein the NLC particle comprises a solid shell of about 1:1 relative to a liquid core.

11. The NLC particle of any preceding claim, wherein the NLC particle comprises about 1:1 cholesterol relative to castor oil.

12. A composition comprising a plurality of NLC particles according to any preceding claim.

13. The composition of claim 12, wherein the composition does not comprise a therapeutically active agent.

14. The composition of any one of claims 12 or 13, wherein the composition is an ophthalmically acceptable composition.

15. The NLC particle of any one of claims 1 to 11, or the composition of any one of claims 12 to 14, for use in treating or preventing a dry eye disorder in a subject.

16. Use of the NLC particle of any one of claims 1-11 or the composition of any one of claims 12-14 in the manufacture of a medicament for treating or preventing a dry eye disorder in a subject.

17. A method of treating or preventing a dry eye disorder in a subject, comprising administering the NLC particle of any one of claims 1 to 11 or the composition of any one of claims 12 to 14 to an eye of the subject.

18. The method of claim 17, wherein the administration is topical to the surface of the eye or eyelid.

19. An eye drop dispenser or eye wash device comprising the NLC particle of any one of claims 1 to 11 or the composition of any one of claims 12 to 14.

Technical Field

The present invention relates to Nanostructured Lipid Carrier (NLC) particles for ocular administration, and related compositions, uses and treatments.

Background

Dry eye (keratoconjunctivitis sicca) is a common disease with a variety of underlying causes. It is characterized by a long-term lack of adequate lubrication and moisture on the surface of the eye, resulting in tear film breakdown. The three main components of the tear film are: oil, water and mucin, each component being provided by a different gland present on or around the eye. An imbalance between these three components, for example, due to dysfunction of one or more glands, leads to symptoms of tear film insufficiency and dry eye.

One of the major causes of dry eye is Meibomian Gland Dysfunction (MGD). The meibomian glands are small glands found in the upper and lower eyelids and function to secrete oil onto the surface of the eye, thereby reducing the evaporation rate of tears. Meibomian gland dysfunction, through obstruction or other abnormalities, results in decreased secretion of oil and subsequent increased rate of tear evaporation, leading to dry eye.

One of the major challenges facing pharmacologists and formulation scientists is ocular administration. The eye is complex, has a unique physiological and anatomical structure, and is considered to be a protective organ that is highly unaffected by foreign bodies. The eye has a lacrimation mechanism, which can hinder the bioavailability of formulations such as eye drops. Structural disorders of the eye allow only less than 5% of a typical formulation to enter the eye. Therefore, formulation scientists have a challenge to develop eye drops that can overcome this obstacle.

There are several eye drops based on artificial tears on the market. The artificial tears have relieving effect on xerophthalmia. These are lubricious eye drops used to treat dryness and irritation associated with insufficient tear secretion in keratoconjunctivitis sicca. One example of a non-drug emulsion based lubricious eye drop is RefreshAs described above, most of eye drops have low bioavailability due to lacrimation, resulting in a decrease in therapeutic effect. To overcome this problem, frequent dosing is required to achieve therapeutic concentrations.

Commercial emulsions also face instability such as coalescence, flocculation and creaming. To prevent this instability, they are usually formulated with surfactants to improve the kinetic stability of the emulsion. However, the addition of surfactants to emulsions is associated with cytotoxicity. In particular, suspension-based formulations are reported to exhibit disadvantages such as additive toxicity and blurred vision. To overcome these challenges, safer and more effective artificial tear compositions are needed.

Disclosure of Invention

According to a first aspect of the present invention, there is provided a Nanostructured Lipid Carrier (NLC) particle, wherein the nanostructured lipid carrier particle comprises:

(i) a solid shell comprising a solid lipid, wherein the solid lipid comprises cholesterol; and

(ii) a liquid core comprising liquid lipids, wherein the liquid lipids comprise triglycerides.

The NLC particles according to the invention are advantageously non-toxic, biocompatible and show a favourable mucoadhesion, which leads to an increased residence time of the NLC particles on the cornea. Triglycerides, such as castor oil or oils like the triglyceride component, may advantageously provide anti-inflammatory properties. Advantageously, it can act as a penetration enhancer, aiding the transcellular transport of NLC particles according to the invention to the cornea. Triglycerides, such as castor oil or oils like the triglyceride component, can also reduce tear evaporation from the eye and, due to their anti-inflammatory effects, can be used to treat dry eye disorders such as meibomian gland dysfunction. Since the cell membrane consists of a lipid bilayer, the use of cholesterol as a solid lipid component will advantageously provide better diffusion of NLCs through the cell membrane.

In one embodiment, the triglyceride comprises a castor oil triglyceride. In particular, the liquid core may comprise or consist of castor oil. In another embodiment, the liquid core may comprise or consist of a castor oil triglyceride or equivalent triglyceride having a fatty acid content substantially similar to that of a castor oil triglyceride.

In one embodiment, the triglyceride comprises omega-3-fatty acids. In particular, the liquid core may comprise or consist of omega-3-fatty acids. In another embodiment, the liquid core may comprise or consist of omega-3-fatty acid triglycerides or equivalent triglycerides with a fatty acid content substantially similar to omega-3-fatty acid triglycerides.

In one embodiment, the triglyceride comprises ricinoleic acid as the major fatty acid component. In one embodiment, the triglyceride comprises at least 85% ricinoleic acid. Triglycerides may include ricinoleic acid, oleic acid and linoleic acid. In one embodiment, the triglyceride comprises from about 85% to 95% ricinoleic acid, from about 2% to 6% oleic acid, and from about 1% to 5% linoleic acid. Additionally or alternatively, the triglycerides may include ricinoleic acid, oleic acid, linoleic acid, alpha-linolenic acid, stearic acid, palmitic acid, and dihydroxystearic acid.

In one embodiment, the NLC particles according to the present invention are suitable for use in the treatment or prevention of dry eye disorders. The dry eye disorder may include a condition selected from dry eye (keratoconjunctivitis sicca); conjunctivitis; keratitis; uveitis; scleritis; episcleritis; blepharitis; and iritis; or any combination thereof. In one embodiment, the therapeutic agent is suitable for treating or preventing dry eye.

In one embodiment, the NLC particles do not encapsulate or comprise a therapeutic agent. In one embodiment, the NLC particles do not encapsulate or comprise a therapeutic agent other than a liquid lipid (e.g., a triglyceride, which itself may be considered to have therapeutic properties, such as anti-inflammatory properties). In another embodiment, the NLC particles do not encapsulate or comprise a therapeutically effective amount of a therapeutic agent. In another embodiment, the NLC particles do not encapsulate or comprise a steroid, such as dexamethasone. Additionally or alternatively, the NLC particles do not encapsulate or contain steroids or antibiotics. Additionally or alternatively, the NLC particles do not encapsulate or comprise a drug selected from one or more or any of the following: anticancer drugs (e.g. docetaxel or irinotecan), antifungal drugs (e.g. amphotericin B), nutraceutical/agents (e.g. curcumin, resveratrol, honokiol/magnolol or quercetin). Additionally or alternatively, the NLC particles may not encapsulate or comprise chalcone, or a therapeutically effective amount of chalcone. In one embodiment, the chalcone comprises Isoliquiritigenin (ILTG). In one embodiment, the NLC particles do not encapsulate or contain an anti-cancer drug.

In one embodiment, the NLC particles are not liposomes. The NLC particles may not encapsulate or comprise lecithin and/or vitamin a.

The nanostructured lipid carrier particles can be nanoparticle sized. In one embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 1000 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 900 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 800 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 700 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 600 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 500 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 400 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 300 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 200 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 100 nm. In another embodiment, the nanostructured lipid carrier particles have a maximum diameter of less than about 50 nm. In another embodiment, the nanostructured lipid carrier particle has a maximum diameter between about 5nm and about 1000 nm. In another embodiment, the nanostructured lipid carrier particle has a maximum diameter between about 5nm and about 800 nm. In another embodiment, the nanostructured lipid carrier particle has a maximum diameter between about 5nm and about 500 nm. In another embodiment, the nanostructured lipid carrier particle has a maximum diameter between about 5nm and about 100 nm. In another embodiment, the nanostructured lipid carrier particle has a maximum diameter between about 5nm and about 50 nm. In another embodiment, the nanostructured lipid carrier particle has a maximum diameter between about 5nm and about 30 nm. In another embodiment, the nanostructured lipid carrier particle has a maximum diameter between about 5nm and about 21 nm. In another embodiment, the nanostructured lipid carrier particle may have a maximum diameter of about 304 nm. The nanostructured lipid carrier particles can be substantially spherical in shape.

In one embodiment, the NLC particles comprise a solid shell of about 1:1 relative to the liquid core. The percentage v/w of the liquid core of the NLC particles can be the average in the NLC population.

In one embodiment, the NLC particles comprise about 1:1 cholesterol relative to castor oil. Optionally, the NLC particles comprise about 1:0.5 cholesterol relative to castor oil. Optionally, the NLC particles comprise about 1:1.5 cholesterol relative to castor oil. In another embodiment, the NLC particles comprise cholesterol at about 1:0.5-1.5 relative to castor oil. In another embodiment, the NLC particles comprise cholesterol in a ratio of about 1:0.8-1.2 relative to castor oil. The percentage v/w of the liquid core of the NLC particles can be the average in the NLC population.

According to another aspect of the present invention, there is provided a composition comprising a plurality of NLC particles according to the invention herein.

The composition may be a pharmaceutically acceptable composition. The composition may be an ophthalmically acceptable composition. For example, NLC particles can be provided in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be an ophthalmically acceptable carrier. The composition may be suitable for topical administration to the eye. In one embodiment, the composition is an ophthalmic composition. Ophthalmic compositions are understood to be sterile, liquid, semi-solid or solid formulations which may or may not contain one or more active pharmaceutical ingredients intended for application to the eye or eyelid (i.e. the anti-inflammatory castor oil described herein).

The composition may be in the form of NLC particles suspended in a gel, lotion, cream, ointment, or solution such as an aqueous solution. In one embodiment, the composition is in the form of an eye drop formulation.

The composition may comprise one or more ophthalmically acceptable ingredients selected from the group consisting of: water; brine; salt; a buffering agent; a demulcent; a humectant; a tackifier; a tonicity adjusting agent; cellulose derivatives, such as sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose or methylcellulose; (ii) dextran 70; gelatin; a polyol; glycerol; polyethylene glycols, such as PEG300 or PEG 400; polysorbate 80; propylene glycol; polyvinyl alcohol; and povidone (polyvinylpyrrolidone); and combinations thereof.

The demulcent may comprise or consist of: cellulose derivatives, glycerin, polyvinyl alcohol, polyvinyl pyrrolidone, cellulose derivatives, polyethylene glycol, or combinations thereof.

In one embodiment, the carrier of the NLC particles in the composition comprises substantially no surfactant, such as polysorbate/tween 80. In one embodiment, the carrier of the NLC particles in the composition does not comprise a physiologically relevant concentration of surfactant, such as polysorbate/tween 80. In one embodiment, the carrier of the NLC particles in the composition comprises no more than 0.05% or no more than 1% of a surfactant, such as polysorbate/tween 80. In one embodiment, the carrier of the NLC particles in the composition comprises no more than 1.5% surfactant, such as polysorbate/tween 80.

According to another aspect of the present invention there is provided a composition according to the invention herein for use in the treatment or prevention of a dry eye disorder in a subject.

According to another aspect of the present invention there is provided a use of a composition according to the invention herein in the manufacture of a medicament for treating or preventing a dry eye disorder in a subject.

According to another aspect of the present invention, there is provided a method of treating or preventing a dry eye disorder in a subject, comprising administering to the eye of the subject a composition according to the present invention.

Administration may be topically to the surface of the eye or to the eyelid.

The subject may be a mammal. In one embodiment, the subject is a human subject. The subject may need to be treated for a dry eye disorder or may be at risk of developing a dry eye disorder.

Treatment or prevention may include a single administration or repeated administrations. Administration may be once daily, or at least once daily. Administration may be once every 2 days. Administration may be once every 1 to 7 days. Administration may be twice daily. The administration may be more than twice daily. Administration may be three or more times a day.

Dry eye disorders may be selected from dry eye disorders (keratoconjunctivitis sicca); conjunctivitis; keratitis; uveitis; scleritis; episcleritis; blepharitis; and iritis; or a combination thereof. The disorder may be acute or chronic.

In another aspect of the present invention, there is provided an eye drop dispenser or eye wash device comprising a composition according to the present invention.

The eye drop dispenser may also be referred to as an eye drop applicator. A typical eye drop dispenser includes a reservoir for the composition and an outlet for the composition. The outlet may taper towards the distal end with the outlet aperture at the tip/distal end. The dispenser may be arranged to be sealed, for example with a lid. The eye drop dispenser may optionally include a syringe device.

The skilled person will be familiar with the term "Nanostructured Lipid Carrier (NLC)" where it is used for artificial tears composed of solid and liquid lipids as the core matrix.

In the context of a solid shell, the term "solid" is understood to mean a solid at room temperature (i.e. about 24 ℃), i.e. the shell is solid at room temperature. In the context of a liquid core, the term "liquid" is understood to mean a liquid at room temperature (i.e. about 24 ℃), i.e. the core is a liquid at room temperature. In one embodiment, the solid shell of the NLC particles is solid at body temperature (e.g., 37 ℃).

In the context of solid lipids, the term "solid" is understood to mean that the composition of lipids is solid at room temperature (i.e. about 24 ℃), i.e. the lipids are present as a solid at room temperature. In one embodiment, the solid lipid forms a solid at body temperature (e.g., 37 ℃). In the context of liquid lipids, the term "liquid" is understood to mean that the composition of lipids is liquid at room temperature (i.e. about 24 ℃), i.e. the lipids are present as a liquid at room temperature.

The term "prevention" refers to the avoidance of a disorder or the protective treatment of a disorder. Prevention may include reducing the risk of the disorder, reducing the risk of infection, the risk of transmission and/or progression, or reducing the severity of the disorder.

The term "treatment" refers to a cure of a condition or disease, alleviation of symptoms, or lessening of the severity of a disorder or symptoms of a disorder.

The term "dry eye disorder" as used herein is to be understood as referring to any condition that is typically symptomatic of dry eye, such as a feeling of dryness, gritty or aching, itching, stinging or tiredness of the eye that is worsening throughout the day, possibly accompanied by pain and/or redness. For example, dry eye (keratoconjunctivitis sicca), meibomian gland dysfunction, or dry eye associated with the use of contact lenses or certain drugs.

The term "therapeutic agent", "therapeutically active" or "active agent" as used herein should be understood to mean any small molecule or biologic drug that: it is configured to bind directly to a cell, cellular component, cell membrane, nucleic acid, or protein (e.g., an enzyme) and is intended to activate or inhibit a biological pathway or other cellular process. In an embodiment, the solid lipid and/or the liquid lipid (e.g., cholesterol and/or triglycerides, respectively) may not be considered a small molecule or a biopharmaceutical/therapeutic agent. In another embodiment, solid lipids and/or liquid lipids (e.g., cholesterol and/or triglycerides, respectively) may be considered to have a therapeutic effect, but the NLC may not comprise other drugs/therapeutic agents.

The skilled person will appreciate that optional features of an embodiment or aspect of the invention may be applied to other embodiments or aspects of the invention where appropriate.

Drawings

Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

FIG. 1. cytotoxicity study of cholesterol castor oil-NLC (CHCAO-NLC) using MTT assay (colorimetric assay for assessing metabolic activity of cells).

FIG. 2 cellular uptake studies of CHCAO-NLC using fluorescence microscopy.

Figure 3 in vitro mucoadhesion studies using fluorescence microscopy. Porcine eye corneas were incubated for 4 hours using CHCAO-NLC and then examined at 10-fold magnification. (a) Control in DIC mode; (b) control on FITC pattern; (c) CHCAO-NLC processed in DIC mode; (d) CHCAO-NLC in FITC mode.

FIG. 4 MMP-9 labeling study of CHCAO-NLC. C-negative control; a positive control of C + L-LPS; c + L + D0.1 or 0.5-cells with different concentrations (. mu.g/ml) of CHCAO-NLC.

Detailed Description

Example 1-dimensional measurement and zeta potential measurement:

particle size and zeta potential NLC are characterized by DLS. Characterization showed that the sample with a cholesterol-castor oil nanostructured lipid carrier (CHCACO-NLC) ratio of 1:1 was most stable compared to other ratios. The size and zeta potential of CHCAO-NLC are shown in Table 1. Sample particle size (nm) Zeta potential (mV)

Table 1 size and zeta potential of CHCAO-NLC.

Sample name	Particle size (nm)	ZETA potential (mV)
			CHCAO1:1	304	-8.5

Example 2-cytotoxicity assessment of prepared CHCACO-NLC:

cytotoxicity of CHCAO-NLC was performed on Human Corneal Epithelial Cells (HCEC). Cells with an initial density of 10,000 cells/well were seeded in a 96-well plate, and then cultured in DMEM medium containing 10% FBS for 24 hours. The cells were then treated with different concentrations of CHCAO-NLC and the CHCAO-NLC treated cells were incubated in a humidified environment of 5% CO2 at 37 ℃ for 4 hours. After 4 hours of culture, the medium was replaced with fresh medium and the cells were maintained for an additional 20 hours. After a specified time, MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) reagent was added to each well and the cells were cultured for an additional 4 hours. The medium in each well was replaced with 200 μ L DMSO to dissolve the formazan crystals. The absorbance (o.d.) was recorded with a microplate reader. Cell viability was calculated by using the following equation:

cell viability (%) [ o.d. (test)/o.d. (control) ] x100

Wherein o.d. (test) and o.d. (control) are absorbance values of cells cultured with and without NLC, respectively.

Cytotoxicity studies showed that the prepared 1:1 ratio of CHCAO-NLC was non-toxic at the given concentrations in FIG. 1.

Example 3 cellular uptake study

The coumarin-6 labeled CHCAO-NLC was further examined for in vitro cellular uptake using fluorescence microscopy. Cells were plated at 5X10 per dish⁴The density of individual cells was seeded in confocal imaging dishes. Coumarin-6 labeled CHCAO-NLC was prepared and exposed to HCEC cells in culture. After 4 hours of incubation at 37 ℃, CHCAO-NLC treated medium was removed and the cells were washed. Further cellular uptake studies were performed under a fluorescent microscope. The data show that NLC are internalized in the cell and coumarin-6 encapsulated in CHCAO-NLC is successfully released into the cells of HCEC cellsIt is medium in nature. Indicating that dexamethasone encapsulated in CHCAO-NLC will be released in HCEC cells (FIG. 2).

Example 4 mucoadhesion Studies

Mucosal adhesion studies were performed ex vivo with porcine cornea. The coumarin-6 labeled CHCAO-NLC was further examined on the mucosal adhesion studies on porcine cornea using fluorescence microscopy. The porcine cornea was excised and placed in a confocal imaging dish containing culture medium. Coumarin-6 labeled CHCAO-NLC was prepared and exposed to the cornea in culture. After 4 hours of incubation at 37 ℃, CHCAO-NLC treated medium was removed and the cornea washed. Further mucoadhesion studies were performed under a fluorescent microscope. Mucoadhesion studies have shown that CHCAO-NLC has mucoadhesion. Due to the adherence of CHCAO-NLC particles to the cornea (FIG. 3).

Example 5 marker Studies

Marker studies were performed on the MMP-9 protein. Mix 5x10⁴Individual cells were seeded in 6-well plates containing DMEM medium. When confluence reached 70%, cells were transfected with lipopolysaccharide and cultured in a humidified environment of 5% CO2 at 37 ℃ for 24 hours. After 24 hours, the medium was replaced with fresh medium, cells were transfected with different concentrations of CHCAO-NLC and cultured for 24 hours. Proteins were extracted from the cells after 24 hours and used in enzyme-linked immunosorbent assay (Elisa). Elisa was performed to measure MMP-9 concentration. A decrease in MMP-9 marker concentration was observed (FIG. 4).