NANOTECHNOLOGY-BASED DELIVERY SYSTEM OF BERGAMOT ESSENTIAL OIL, METHOD OF PREPARATION OF THE SYSTEM AND USES THEREOF

Abstract

The present invention concerns a nanotechnology-based delivery system of bergamot essential oil (BEO) that are α-tocopheryl stearate-Solid Lipid Nanostructures (α-TFS-SLNs) loaded with bergamot essential oil without psoralens (BEO-BF). The present invention concerns also a method of preparation and uses of said α-tocopheryl stearate-Solid Lipid Nanostructures (α-TFS-SLNs) loaded with bergamot essential oil (BEO-BF).

Claims

1. Solid lipid nanoparticles, wherein said nanoparticles are α-tocopheryl stearate based and comprise bergamot essential oil, preferably free of psoralens.

2. The solid lipid nanoparticles according to claim 1, wherein said solid lipid nanoparticles have size of 444.03nm±60.07.

3. The solid lipid nanoparticles according to claim 1, wherein said bergamot essential oil comprises limonene, linalool and linalyl acetate.

4. A pharmaceutical composition comprising the solid lipid nanoparticles according to claim 1, as active principle, in association with one or more excipients and/or adjuvants.

5. The pharmaceutical composition according to claim 4, said pharmaceutical composition being in the form of a cream or gel.

6. (canceled)

7. A method of use in the treatment of acute and chronic pain, such as the neuropathic type of pain or pain occurring in the course of cancer or pain occurring in the course of chronic neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease, comprising administering to a subject the solid lipid nanoparticles according to claim 1.

8. A method of use in the prevention or treatment of behavioural and psychiatric symptoms of dementia, or of behavioural or mood disturbances emerging from stressful conditions, such as itch, comprising administering to a subject the solid lipid nanoparticles according to claim 1.

9. A method for obtaining the solid lipid nanoparticles according to claim 1, said method comprising or consisting of: a) mixing αopheryl stearate in the presence of bergamot essential oil by heating, for example bergamot essential oil is in a percentage of 10% in comparison to αopheryl stearate; b) adding a water solution of a one or more emulsifiers, such as polysorbate 20, polysorbate 60, soy phosphatidylcholine, and sodium taurodeoxycholate, preferably sodium taurodeoxycholate, one or more co-emulsifiers, such as sodium monooctylphosphate, n-butanol, preferably butanol, water, and optionally one or more surfactants, such as Tween 20, in order to obtain a microemulsion; and c) dispersing the microemulsion in cold water under agitation, preferably in a ratio microemulsion:cold water of 1:20.

10. The pharmaceutical composition according to claim 4, wherein said solid lipid nanoparticles have size of 444.03nm±60.07.

11. The method according to claim 7, wherein said solid lipid nanoparticles have size of 444.03nm±60.07.

12. A method of use in the treatment of acute and chronic pain, such as the neuropathic type of pain or pain occurring in the course of cancer or pain occurring in the course of chronic neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease, comprising administering to a subject the pharmaceutical composition according to claim 4.

13. A method of use in the prevention or treatment of behavioural and psychiatric symptoms of dementia, or of behavioural or mood disturbances emerging from stressful conditions, such as itch, comprising administering to a subject the pharmaceutical composition according to claim 4.

Description

[0063] The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the enclosed drawings, wherein:

[0064] FIG. 1 shows Photomicrography of SLNs by Trasmission Electron Microscopy. Ultrastructure of the modified Solid Lipid Nanoparticles (SLN) loaded with bergamot essential oil without psoralens (BEO-BF) is shown. At six months the percentage of linalool, linalyl acetate and limonene SLNs containing was declined of 17.7+2.52% respect to that present into SLNs analysed at time zero (linalool=10.6 mg; linalyl acetate=12.1 mg and limonene=14.1 mg, respectively).

[0065] FIG. 2 shows the Effects of NDS with BEO-BF on capsacin-induced nociceptive behavior in mice. In particular, it is shown that, using the lot n° 1/2016 of the pharmaceutical cream preparation, BEO-BF encapsulated into modified SLN (cream) NDS is endowed with antinociceptive properties in the capsaicin test in mice. NDS BEO-BF cream (1 mg) (test) or empty (control) (1 mg) were applied to the plantar surface of the right hindpaw in mice (n=8) 30 min before capsaicin injection and cumulative time (sec) spent in licking/biting behaviour was monitored for a 5 min session.

[0066] FIG. 3 shows the Effects of NDS with BEO-BF on formalin-induced nociceptive behavior in mice. In particular, it is shown that, using the lot n° 1/2016 of the pharmaceutical cream preparation, BEO-BF encapsulated into modified SLN (cream) NDS is endowed with antinociceptive properties in the formalin test in mice. NDS BEO-BF cream (1 mg) (test) or empty (control) (1 mg) were applied to the plantar surface of the right hindpaw in mice (n=8) 30 min before formalin test and cumulative time (sec) spent in licking/biting behaviour was monitored for a 30 min session.

[0067] FIG. 4 shows the Effects of NDS with BEO-BF on neuropathic pain in mice with partial sciatic nerve ligation. In particular, it is shown that, using the lot n° 1/2016 of the pharmaceutical cream preparation, BEO-BF encapsulated into modified SLN (cream) NDS is endowed with antiallodynic properties in the partial sciatic nerve ligation (PSNL) test model in mice. NDS BEO-BF cream (1 mg) (test) or empty (control) NDS cream (1 mg) were applied to the plantar surface of the right hindpaw in mice (n=10) 30 min before sensitization measurement. BEO (test) and control have been administered on day 7 after surgery and mechanical allodynia has been measured as withdrawals of the foot upon application of the Von Frey filament (3.0 g)and repeated in ten sessions (modified from Kuwahata et al., 2013).

[0068] FIG. 5 shows the Effects of BEO cream on itching in mice. In particular, it is shown that, using the lot n° 1/2016 of the pharmaceutical cream preparation, BEO-BF encapsulated into modified SLN (cream) NDS is endowed with anti-itching properties in the test model provided by intradermal (i.d.) administration of 4 methyl-histamine (200 μg/50 μl, i.d.) in rear region of the neck of mice. NDS BEO-BF cream (1 mg) (test) or empty NDS cream (1 mg) (control) were applied on the shaved skin of the rear region of the neck 30 min before (FIG. 5A; n=8 mice) or immediately (0 min) before (FIG. 5B, n=8 mice) 4 methyl-histamine intradermal (i.d) administration in the shaved skin region and scratching behaviour was measured for 30 min following administration.

EXAMPLE 1

Preparation of α-TFS-SLNs and Related Cream for Skin Application

[0069] Reagents

[0070] All solvents were of analytical grade and purchased from Sigma-Aldrich (Sigma Chemical Co., St. Louis, Mo., USA): tetrahydrofuran (THF), chloroform (CHCl3), n-hexane, ethyl acetate, dimethyl sulphoxide (DMSO), isooctane, 1-butanol, α-linolenic acid (PM=278.43 g/mol), α-tocopherol (PM=430.72 g/mol), biliary salt of the taurodeossicolic acid, Tween 20, dicyclohexylcarbonymide (DCC).

[0071] Synthesis of Alfa-Tocopheryl Stearate (α-TFS)

[0072] α-tocopheryl stearate (α-TFS) was synthesized according to the procedure described in the literature (Taniguchi et al., 1998). Briefly α-tocopherol (1.0 g, 5.15 mmol), p-toluensulfonic acid (0.07 g, 0.34 mmol) and stearyl alcohol (0.7 g, 5.4 mmol) were added to 10 ml dry toluene under stirring and N.sub.2 at room temperature. When the addition was complete, the solution was stirred under N.sub.2 for 12 h at 110° C. After cooling at room temperature, the solvent was evaporated under reduced pressure, the residue was treated with 15 ml water and the aqueous phase was extracted with chloroform (4×15 ml). The combined organic phases were collected and dried using natrium sulphate (Na.sub.2SO.sub.4), a common inorganic drying agent that acquire water of hydration when exposed to moist air or a wet solution. Afterwards the solvent was removed by rotary evaporation to give yellow-colored alpha tocopheryl stearate (α-TFS) and purified by Merck silica gel (60-230 mesh) column chromatography, using ethylacetate/n-hexane85/15(v/v) as eluent. The solvent of eluated ester was evaporated under reduced pressure. The α-TFS was identified by TLC, UV and 1H-NMR analyses (Bernards and Lewis, 1992; Kawanishi et al.,1990; Taniguchi et al., 1998).

[0073] Preparation of α-TFS-SLNs

[0074] α-tocopheryl stearate-Solid Lipid Nanoparticles (α-TFS-SLNs) were prepared by a microemulsion technique at moderate temperature (Gasco, 1997). In particular, α-tocopheryl stearate-Solid Lipid Nanoparticles (α-TFS-SLNs) were prepared by a microemulsion technique. Briefly, α-tocopheryl stearate (α-TFS, 142 mg, 0,201 mmol) in the absence and/or in the presence of BEO (National origin: Italy) (40 mg, 10% of α-TFS, 0.0201 mmol) was mixed at a temperature ranging from 60 to 65° C. A warm water (0.21 mg, 6% of α-TFS 0.0012 mmol) solution of sodium taurodeoxycholate (25.33 mg, 30% α-TFS, 0.061 mmol), butanol (0.3 mg, 2% of α-TFS, 0.0041 mmol) and Tween 20 (75 mg, 30% of α-TFS, 0.061 mmol) was then added to obtain an optically transparent system. Sodium taurodeoxycholate and Tween 20 act as emulsifiers and butanol as co-emulsifier. The warm microemulsion was immediately dispersed in cold water (2° C.) under high-speed homogenation (Model SL2, Silverson, Chesham Bucks, England) at 8000 rpm for 30 min (240.000 g in 30 min). The volume ratio of warm microemulsion to cold water was 1:20. The α-TFS-SLN dispersions were washed twice using an Amicon TCF2A ultrafiltration system (Amicon Grace, Beverley, Mass. USA; membrane Amicon Diaflo YM 100).

[0075] Size Distribution Analysis

[0076] The size of the α-TFS-SLNs was determined by dynamic light scattering (DLLS) using a 90 Plus Particle Size Analyzer (Brookhaven Instruments Corporation, New York, USA) at 25° C. by measuring the autocorrelation function at 90° scattering angle. Cells were filled with 100 pl of sample solution and diluted to 4 ml with filtered (0.22 μm) water. The polydispersity index (PI) indicating the measure of the distribution of nanoparticle population was also determined (Koppel, 1972). The SLNs showed a diameter equal to 450 nm and a polydispersity index of 0.30.

[0077] Morphology of SLN

[0078] The morphology of the hydrated SLN dispersions was examined using TEM. In particular, a drop of SLN dispersion was applied to a carbon-coated copper grid and left for 1 min to allow some of the particles to adhere to the carbon substrate. The excess of dispersion was removed by adsorbing the drop with a piece of filter paper. A drop of 1% phosphotungstic acid solution was applied and again excess of solution was removed by adsorbing the liquid with the tip of a filter paper and the sample was air-dried. The sample was then observed under a ZEISS EM 900 electron microscope at an accelerating voltage of 80 kV. The image observed is shown in FIG. 1.

[0079] Percentage of BEO-BF Incorporated into α-TFS-SLN

[0080] α-TFS-SLN formulation (1 ml) was diluted to 10 ml with methanol and, to evaluate BEO-BF content, was analyzed by spectrophotometric detection at wavelengths of:

[0081] 281 nm for Linalol,

[0082] 208 nm for a Linalyl acetate, and

[0083] 247 nm for Limonene, respectively.

[0084] The results indicated the presence, in α-TFS-SLN of:

[0085] 27% of Linalol,

[0086] 31% of Linalyl acetate, and

[0087] 36% of Limonene.

[0088] After two and six months of light exposure the above percentages declined by 10% and 18%, respectively, with no further degradation at twelve months, demonstrating that α-TFS-SLNs effectively slow down degradation of BEO-BF.

[0089] Preparation of a Nanocream Delivery System (NDS) Based on α-TFS-SLN Containing BEO-BF (44.227 g)

[0090] The composition of cream based on α-TFS-SLN containing BEO-BF is reported below:

[0091] 37.604 g of purified water suspension of α-TFS-SLN containing BEO-BF,

[0092] 4.42 g of sweet almond oil,

[0093] 0.885 g of polyacrylamide,

[0094] 0.442 g of isoparaffin C13-14,

[0095] 0.111 g of 7-laurate

[0096] 0.774 g of purified water Ph.Eur.,

[0097] 0.028 g of methyl paraben, and

[0098] 0.009 g of propylparaben.

EXAMPLE 2

Prolonged Antinociceptive and Anti-Itching Actions of the BEO-BF Encapsulated in NDS Cream Shown on Animal Experimental Models

[0099] Capsaicin Test

[0100] NDS BEO-BF cream (1 mg) or empty NDS cream (1 mg) were applied to the plantar surface of the right hindpaw of a mice 30 min before capsaicin injection.

[0101] In these experiments, male ddY (SD) mice (Shizuoka Laboratory Center, Japan) weighing 22-26 g, at the time of testing, were used. The mice were individually housed in a colony maintained in a controlled environment (12 h light/dark cycle, room temperature 23° C., 50-60% relative humidity). The animals had free access to food pellets and water. All behavioural experiments took place during the light period between 10:00 and 17:00 h in a quiet room. The animals were tested in randomized order.

[0102] Antinociception was assessed using the capsaicin test as previously described in Sakurada et al. (1992) (Sakurada et al., 1992). To reduce variability, each mouse was acclimatized to an acrylic observation chamber (22.0×15.0×12.5 cm) for approximately 1 h before the injection of capsaicin. The mouse was injected 20 μl of a solution of capsaicin (0.8 μg/paw) beneath the skin of the plantar surface of the right hindpaw using a 50 μl Hamilton microsyringe with a 26-gauge needle as quickly as possible, with only minimal animal restraint. Following capsaicin injection, the animals (n=16) were immediately placed in the test box for a 5-min observation period. Licking/biting behaviour induced by intraplantar injection of capsaicin was observed as an indicator of nociceptive response. The accumulated response time in second spent in licking/biting the capsaicin-injected paw was measured for a period of 5 min immediately after subcutaneous (s.c.) injection of capsaicin.

[0103] Results are shown in FIG. 2.

[0104] Formalin Test

[0105] In the formalin test, male ddY mice (n=16) (Japan SLC, Hamamatsu, Japan) were placed into a transparent cage (22.0 cm×15.0 cm×12.5 cm high) which also served as an observation chamber and were allowed to adapt to their environment for 1 h before testing. After this period, plantar subcutaneous injection of 20 μL formalin (2% in saline) using a microsyringe with 26-gauge needle. BEO and control have been administered 10 min before the injection of formalin. Each mouse was immediately returned to the observation chamber after injection. The recording of the first response (first phase) started immediately and lasted for 10 min (0-10 min). The recording of the late response (late phase) started 10 min after formalin injection and lasted for 20 min (10-30 min). In both phases, licking and biting of the injected hindpaw were defined as a nociceptive response and the total time (s) of the response was measured with a hand-held stop-watch.

[0106] Results are shown in FIG. 3.

[0107] PSNL (Partial Sciatic Nerve Ligation) Test

[0108] Male ddY mice (n=20) (Kyudo Industries, Kumamoto, Japan) were anesthetized by pentobarbital anesthesia (50 mg/kg, i.p.) following the methods of Malmberg and Basbaum (1998) (Malmberg & Basbaum, 1998). BEO and control have been administered on post-operative day 7. The sciatic nerve of the right hindlimb was exposed at high thigh level through a small incision and the distal one third to one half of the dorsal portion of the sciatic nerve was tied with non-absorbable silk thread. The wound was closed with silk thread suture and covered with antibiotic powder. Immediately following surgery, the animals were kept in a soft bag cage with some food inside so that they could feed themselves without having difficulty standing. The wound healed within 1 to 2 days, and the mice with ligated nerves did not present signs of foot clonus or autotomy, but behaved normally.

[0109] The presence of mechanical allodynia (sensitization) was determined by an electronic version of the von Frey test (dynamic plantar anesthesiometer, model 37400; Ugo Basile, Milan, Italy). Animals were allowed to habituate to the testing environment and Plexiglass observation chamber (11.0×17.0×14.0 cm, length×width×height) with a wire mesh floor for approximately 1 h prior to testing. Through the wire mesh floor of the chamber, a servo-controlled mechanical stimulus (a pointed metallic filament) was applied to the midplantar region of the hindpaw. When a trial is initiated, the device raises the filament to touch the foot and progressively increases force until the animal withdraws its foot. The electro-von Frey device is capable of applying a progressively increasing punctate pressure, reaching up to 3.0 g within 5.0 s. The pressure evoking a clear voluntary hindpaw withdrawal response (usually close to 3.0 g) was recorded automatically and taken as the mechanical threshold index. In the time course experiment of PSNL-induced allodynia, baseline responses to mechanical stimulation were obtained before injury and from day 1 to day 35 post-PSNL injury. The effect of NDS BEO-BF or empty NDS on mechanical hypersensitivity was measured 7 days after the surgery when allodynia is fully developed and the pressure applied was 4.0 g. Mechanical paw withdrawal thresholds were evaluated before and after i.pl. injection. Each paw was tested three times to yield a mean value.

[0110] All the testing was performed by a blind observer.

[0111] Results are shown in FIG. 4.

[0112] Itch Test

[0113] The effects of NDS BEO-BF cream on scratching behaviour induced by 4-methyl-histamine administration were studied in the ddY mice (Shizuoka Laboratory Center, Japan). Groups of mice (n=8 per group) were pretreated with NDS BEO-BF cream (1.0 mg/mice) or control cream (1.0 mg/mice) for transdermal application 30 min prior or immediately before intradermal (i.d.) administration of 4 methyl-histamine (200 μg/μl) and the scratching behaviour filmed for 30 min and measured offline by an independent observer. The 4-methyl-histamine is a pharmacological tool used to induce itching behaviour in mice. Latency time is of 10 minutes. It has been observed the occurrence of scratching behaviour in the mice subjected only to 4-methyl-histamine administration. This behaviour, more intense in the first minute of observation, but occurring also from 2 min and 40 seconds, was completely prevented by the topical application of the nanocream delivery system (NDS) based on α-TFS-SLN containing BEO-BF.

[0114] Results are shown in FIG. 5.

EXAMPLE 3

Formulation According to the Present Invention and Method of Preparation Thereof

[0115] Suspension of SLN in purified water FU g 84.75

[0116] sweet almond oil g 10

[0117] butylhydroxytoluene (BHT) 0.05 g

[0118] sepigel g 5 (polyacrylamide 40%, C13-14 isoparaffin 20%, 7-laurate 5%, purified water FU 35%)

[0119] methyl 4-hydroxybenzoate 0.18 g

[0120] propyl 4-hydroxybenzoate 0.02 g

[0121] The pharmaceutical preparation requires the initial nipagine solubilization by magnetic stirrer at a temperature of 60° C., in a quantity of water equal to half of the acqueous phase (39.88 g) present in the formulation. After nipagine solution cooling, the SLN suspension (5 ml) and the rest of the purified water were added to obtain the final acqueous phase weight (84.75 g).

[0122] Solubilizing the BHT in almond oil, pouring the previously prepared solution with a quantity more or less equal to the quantity of oil, adding sepigel and stirring with a glass rod. Continuing with a geometric dilution until all the aqueous suspension has been used up. The emulsion thus obtained is processed in citounguetor for 2 minutes at speed 2. This method has the following advantages: [0123] homogeneously incorporating SLNs without the use of a turboemulsifier which could vary the volume and/or damage the nanoparticles; [0124] not changing pH of the initial solution containing SLN; [0125] Forming a creamy base in cold conditions, a fundamental point for particles integrity.

[0126] On the basis of the previous points the possibility of creams and gels whose preparation is closely related to temperature and heat has been ruled out.

[0127] Another method can be the suspension of SLNs in cold conditions in a basic Pharmacopoeia cream such as cetomacrogol, with the limit of nanoparticle dispersion that may not be homogeneous due to the failure to use a turbine.

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