WO2023062597A1 - Nanosystems for carrying/encapsulating insoluble molecules of plant origin, including rosmarinic acid - Google Patents
Nanosystems for carrying/encapsulating insoluble molecules of plant origin, including rosmarinic acid Download PDFInfo
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- WO2023062597A1 WO2023062597A1 PCT/IB2022/059866 IB2022059866W WO2023062597A1 WO 2023062597 A1 WO2023062597 A1 WO 2023062597A1 IB 2022059866 W IB2022059866 W IB 2022059866W WO 2023062597 A1 WO2023062597 A1 WO 2023062597A1
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- rosmarinic acid
- surfactant
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- DOUMFZQKYFQNTF-MRXNPFEDSA-N rosemarinic acid Natural products C([C@H](C(=O)O)OC(=O)C=CC=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-MRXNPFEDSA-N 0.000 title claims description 111
- TVHVQJFBWRLYOD-UHFFFAOYSA-N rosmarinic acid Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=Cc2ccc(O)c(O)c2)C=O TVHVQJFBWRLYOD-UHFFFAOYSA-N 0.000 title claims description 111
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/12—Aerosols; Foams
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
Definitions
- the present invention relates to novel nanosystems optimized for the encapsulation and carrying of active molecules/substances of plant origin and preparation methods thereof.
- the present invention also relates to the use of these systems for the topical treatment of allergies and/or inflammation especially affecting the nose.
- RA Rosmarinic acid
- Rosmarinic Acid is considered one of the most potent naturally occurring polyphenols.
- RA Rosmarinic Acid
- RA is contained in several plants belonging to the Lamiaceae family such as Perilla frutescens, which is its richest source, but it can also be found in other commonly used plants such as Ocimum basilicum, Origanum vulgare, Salvia officinalis, Melissa officinalis and many others.
- the Applicant following intensive and prolonged research and development activity, in order to solve the above technical problems has developed nanosystems capable of effectively encapsulating and carrying natural substances.
- the Applicant has developed nanoparticles capable of encapsulating a variety of natural substances by making them usable in water-based systems and easy to administer.
- the Applicant has identified compositions comprising said nanosystems that are advantageously formulated for topical administration, preferably for topical nasal administration.
- nanosystems prepared in the present context can be used for topical treatment of inflammatory diseases/disorders.
- nanosystems of the present invention and compositions thereof have no major side effects and can be administered to all categories of subjects, including pregnant or lactating women, or subjects with other comorbidities.
- nanosystems of the present invention and the compositions comprising them are easy to prepare and cost-effective.
- the present invention relates to a solid lipid nanoparticle (NP) comprising or alternatively consisting of:
- the present invention also relates to a method for preparing said nanoparticle according to the present invention, wherein said method comprises the steps of:
- step (II) Mixing said hot system obtained in step (I) with a turbine emulsifier until a homogeneous dispersion is obtained, preferably mixing at a speed from 6000 rpm to 10000 rpm, for example at 9000 rpm;
- step (II) Cooling under stirring the system obtained in step (II) to obtain solid particles with a lipid matrix, dispersed in an aqueous phase.
- the present invention also relates to a nanocrystal comprising or alternatively consisting of:
- the present invention relates to a composition comprising the solid nanoparticle or the nanocrystal according to the present invention, and optionally at least one excipient of pharmacologically acceptable grade.
- the present invention relates to said composition for use in a method of treatment of allergies and/or inflammatory conditions, preferably affecting the nose, or for antimicrobial/antibacterial use.
- FIG. 1 Preparative example scheme of Solid Lipid nanoparticles (SLNs).
- FIG. 1 Preparative example scheme of Nanocrystals (NCs).
- Figures 3-8 Stability tests of the nanosystems object of the present invention.
- Figure 3 Stability at 25 °C of SLNs without active.
- Figure 4 Stability at 4 °C of SLNs without active.
- Figure 5 Stability at 25 °C of SLNs comprising rosmarinic acid.
- Figure 6 Stability at 4 °C of SLNs comprising rosmarinic acid.
- Figure 7 Stability at 25 °C of the NCs.
- Figure 8 Stability at 4 °C of the NCs.
- Figure 9 Interaction times between the SLN-RA sample and mucin.
- Figure 10 Interaction times between the NCs sample and mucin.
- Figure 11 Release profiles of SLNs with SNF and NCs with SNF.
- Figures 12A and 12B Stability studies on FP comprising SLN-RA conducted for a period of 15 days at 25 °C and 4 °C, respectively.
- Figure 13A and 13 B Stability studies on FP comprising NCs conducted for a period of 15 days at 25 °C and 4 °C, respectively.
- Figure 14 Trend over time of the concentration of survived microorganisms in sample N1.
- Figure 15 Trend over time of the concentration of survived microorganisms in sample N2.
- Figure 16 Optical density profile showing the ability of RA to bind to HMGB-1.
- Figure 17 Binding sites of HMGB-1 to rosmarinic acid.
- the present invention relates to innovative nanosystems optimized for encapsulation, carrying and release of natural substances of plant origin, preferably for rosmarinic acid.
- the nanosystems described in the present context are advantageously usable in the medical, pharmaceutical, cosmetic, and nutraceutical fields, and allow safe and effective therapeutic treatments to be obtained through the carrying of natural substances, preferably rosmarinic acid, by overcoming the chemical and physical limitations associated with the substances themselves, while also improving their release profile.
- the nanosystems described in the present context have high stability and allow localized release of the encapsulated active, reducing side effects and being particularly advantageous for topical use.
- nanosystems, and compositions comprising them, according to the present invention advantageously enable efficient carrying of natural active substances.
- nanoparticle comprising at least one surfactant and a substance of plant origin, preferably rosmarinic acid, said substance of plant origin being encapsulated in said surfactant.
- said nanoparticle also comprises a lipid phase that encapsulates/encloses said substance of plant origin.
- said nanoparticle according to the present invention is in the form either of solid lipid nanoparticle (SLN) or nanocrystals (NCs) comprising at least one active substance of plant origin, preferably comprising rosmarinic acid.
- SSN solid lipid nanoparticle
- NCs nanocrystals
- Solid lipid nanoparticles SSNs
- nanocrystals SPNs
- nanocrystals SPNs
- lipid nanoparticle comprising or alternatively consisting of:
- a core comprising or alternatively consisting of (i) a solid-state lipid phase and (ii) at least one active substance of natural origin selected from the group comprising or alternatively consisting of: curcumin, gingerols, rosmarinic acid and mixtures thereof;
- SLNs according to the present invention have sizes in the range from 10 nm to 1000 nm, even more preferably in the range from 20 nm to 600 nm, e.g., in the range from 50 nm to 200 nm, as measured by dynamic light scattering, DLS, measurements.
- SLNs according to the present invention have a weight ratio of lipid component to active substance in the range from 3:1 to 1 :1, e.g. a weight ratio of 2:1.
- the at least one surfactant (b) used for the preparation of SLNs according to the present invention can be a non-toxic surfactant polymer.
- Said surfactant may be a nonionic surfactant, for example, it may be a polysorbate selected from the group consisting of: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80 and mixtures thereof.
- surfactant is polysorbate 80.
- the at least one surfactant (b) is a phospholipid selected from the group comprising or alternatively consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and mixtures thereof.
- phospholipid refers to a lipid whose molecule has a hydrophilic "head” containing a phosphate group, and two hydrophobic "tails” derived from fatty acids, joined by a glycerol molecule.
- said phospholipid used in the present context may be a glycerophospholipid, e.g., it may be selected from the group comprising or alternatively consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lecithin, phosphatidic acid, and mixtures thereof.
- a lecithin such as soy lecithin is used in the present context.
- the at least one surfactant (b) advantageously surrounds and stabilizes the lipid core (a) comprising the active substance of plant origin.
- triglycerides, partial glycerides, fatty acids, steroids and/or waxes and lecithins can be used.
- the lipid phase (i) of SLNs comprises or alternatively consists of Glyceryl monostearate (GMS) and a soy lecithin, e.g., NGM Topcithin (NMG-T) can be used as soy lecithin.
- GMS Glyceryl monostearate
- NMG-T NGM Topcithin
- the lipid phase comprises Glyceryl monostearate (GMS) and/or a soy lecithin (e.g. NGM Topcithin (NGM-T))
- Glyceryl monostearate Glyceryl monostearate
- NGM-T NGM Topcithin
- the NGM Topcithin preferentially used in the present context is a phospholipid belonging to the lecithin family (soy lecithin).
- the use of this component in the lipid phase provides an additional stabilizing effect in addition to that of the surfactant (b).
- SLNs described in the present context can be prepared using different preparation methods known to the person skilled in the art.
- SLNs can be prepared by high-pressure homogenization, hot/cold homogenization, emulsification and solvent evaporation.
- an easy-to-execute method that can be executed on a large scale is used in the present context.
- Said SLNs preparation method comprises the steps of:
- step (II) Mixing said hot system obtained in step (I) with a turbine emulsifier until a homogeneous dispersion is obtained, preferably mixing at a speed from 6000 rpm to 10000 rpm, for example at 9000 rpm;
- step (III) Cooling under stirring, e.g. under a turbine emulsifier, the system obtained in step (II) to obtain solid particles with a lipid matrix, dispersed in an aqueous phase.
- Said preparation method of SLNs may also comprise a step of:
- step (la) Mixing the solubilized active substance in an organic solvent with the hot lipid phase.
- the active substance is rosmarinic acid
- rosmarinic acid can be solubilized in acetone in a step (la). Since acetone is a volatile solvent, it is easily removed from the mixture obtained in step (la).
- the lipid phase obtained in step (la) can be used in step (I) of hot addition and dispersion of said lipid phase with an aqueous phase.
- step (I) is performed at a temperature from 60 °C to 95 °C, preferably at a temperature from 75 °C to 99 °C, e.g. at 90 °C.
- step (I) is performed using sterile disposable syringes.
- the lipid phase comprising the active substance of plant origin, e.g., rosmarinic acid, is heated and inserted into syringes that are then used for the hot addition of the lipid phase to the aqueous phase (step I).
- SLNs according to the present invention have several advantages, including the ability to carry and entrap a high amount of active substance of natural origin, said active substance being preferably rosmarinic acid.
- SLNs according to the present invention are stable systems that are well tolerated by the body and do not pose toxicity risks.
- the SLNs described in the present context allow for a controlled and localized release of the active substance, particularly rosmarinic acid.
- SLNs according to the present invention advantageously enable improved targeting of rosmarinic acid.
- SLNs according to the present invention have high stability and bioavailability.
- a preferred embodiment refers to a solid lipid nanoparticle comprising (a) a core comprising (i) a solid- state lipid phase and (ii) rosmarinic acid,
- a preferred embodiment refers to a solid lipid nanoparticle comprising (a) a core comprising (i) a solid- state lipid phase and (ii) rosmarinic acid, (b) at least one surfactant, wherein said lipid phase comprises a mixture of Glycerylmonostearate (GMS) and NGM Topcithin (NGM- T), and wherein said surfactant (b) is a polysorbate, preferably it is polysorbate 80.
- GMS Glycerylmonostearate
- NGM- T NGM Topcithin
- the SLNs described in the present context can be advantageously dispersed in an aqueous phase.
- the aqueous phase may comprise surfactant (b).
- the pH of the aqueous dispersion comprising SLNs can be adjusted by adding a base, or an acid, in order to obtain the suitable pH for the desired application.
- the pH is adjusted in the range from 3.5 to 6.8. This pH is particularly advantageous in applications for nasal use. Samples have shown high stability in this pH range.
- SLNs according to the present invention are prepared using an amount in mg/ml of lipid phase ranging from 30 mg/ml to 5 mg/ml, even more preferably ranging from 25 mg/ml to 10 mg/ml, e.g. an amount of 18 mg/ml.
- SLNs according to the present invention are prepared using an amount in mg/ml of surfactant ranging from 20 mg/ml to 5 mg/ml, even more preferably ranging from 15 mg/ml to 7 mg/ml, e.g. an amount of 10 mg/ml.
- SLNs according to the present invention are prepared using an amount in mg/ml of active substance of plant origin ranging from 20 mg/ml to 5 mg/ml, even more preferably ranging from 15 mg/ml to 7 mg/ml, e.g. an amount of 10 mg/ml.
- NANOCRYSTALS NANOCRYSTALS
- N-nanocrystals refers to “carrier-free” colloidal drug delivery systems.
- active substances of plant origin preferably rosmarinic acid
- surfactants or surfactant polymers are synthesized in the form of nanocrystals stabilized by surfactants or surfactant polymers.
- nanocrystal comprising or alternatively consisting of (a) an active substance of plant origin and (b) at least one surfactant.
- the present invention also relates to nanocrystals stabilized by at least one surfactant or surfactant polymer stably dispersed in an aqueous system.
- aqueous dispersion comprising nanocrystals comprising or alternatively consisting of (a) an active substance of plant origin, e.g., rosmarinic acid, and (b) at least one surfactant.
- aqueous dispersion comprising nanocrystals comprising or alternatively consisting of:
- rosmarinic acid NCs according to the present invention allow effective carrying of the active substance, the structure of the nanocrystal being entirely composed of that active substance. They thus enable a sufficiently high therapeutic concentration to be achieved for the desired effect.
- NCs in a nano-suspension form, have an average size in the range from 10 nm to 800 nm, even more preferably in the range from 10 nm to 500 nm, e.g, in the range from 10 to 100 nm, as measured by dynamic light scattering, DLS, measurements.
- NCs according to the present invention can occur in crystalline, semi-crystalline or amorphous form.
- nanocrystal NCs described in the present context can be synthesized using techniques known to the person skilled in the art, such as top-down or bottom-up synthesis techniques.
- the former considered high-energy methods, such as dry/wet milling or high-pressure homogenization, involve particle diameter reduction, starting from a large size crystal.
- the latter on the other hand, involve low-energy methods, such as spray drying or nanoprecipitation, based on the aggregation of crystalline or amorphous particles.
- a preferred synthesis method in the present context is nanoprecipitation, which consist of the rapid addition of a solvent (in which the active substance is solubilized), to the antisolvent, resulting in the formation of a precipitate of active.
- the at least one surfactant (b) used to stabilize NCs according to the present invention can be selected from the group comprising or alternatively consisting of: polysorbate 20, polysorbate 80, a polaxamer, and mixtures thereof.
- a polaxamer can be used, for example, Lutrol F68, which is commercially avilable.
- the at least one surfactant (b) is D-o-Tocopheryl polyethylene glycol 1000 succinate, also known as vitamin E TPGS.
- vitamin E TPGS to stabilize NCs according to the present invention enables NCs of rosmarinic acid to be obtained with high stability.
- This substance formed by esterification of vitamin E succinate with polyethylene glycol 1000, has been extensively studied for its emulsifying, dispersing, gelling and solubilizing effects on poorly water-soluble substances.
- Vitamin E TPGS is a derivative of vitamin E, which has antioxidant properties similar to those of tocopherol. It has been used in the past as an excipient to overcome multidrug resistance (MDR) and has been approved by the U.S. Food and Drug Administration (FDA) as a pharmaceutically safe adjuvant.
- MDR multidrug resistance
- FDA U.S. Food and Drug Administration
- NCs according to the present invention make it possible to increase the dissolution rate of the active substance of plant origin, preferably rosmarinic acid, ameliorating issues related to low bioavailability and reduced permeation of the latter.
- NCs according to the present invention are considered low-risk and readily biodegradable, as well as widely adhesive to membranes/mucous membranes. Finally, these systems allow the use of low concentrations of excipients thus reducing excipient-related toxic effects.
- the nanocrystals NCs according to the present invention can be prepared by the following method comprising the steps of:
- step (iii) Adding the solution prepared in step (i) to the antisolvent solution prepared in step (i), magnetically stirring the sample during addition;
- step (iv) Heating the sample in step (iii) to help the evaporation of the organic solvent and obtaining the NCs by precipitation.
- NCs can be redispersed in demineralized water, and the pH can be adjusted according to application needs.
- Step (i) of the NCs preparation method according to the present invention can be carried out at a temperature from 90 °C to 50 °C, preferably from 80 °C to 65 °C, e.g. at 70 °C.
- Step (iv) of the NCs preparation method according to the present invention can be carried out at a temperature from 90 °C to 50 °C, preferably from 75 °C to 55 °C, e.g. at 60 °C.
- Step (i) of preparing an acidified aqueous solution can be carried out by using a hydrochloric acid solution e.g. acidification by adding a 20% wA/ hydrochloric acid solution until the desired pH is obtained, e.g. pH equal to 1.
- a hydrochloric acid solution e.g. acidification by adding a 20% wA/ hydrochloric acid solution until the desired pH is obtained, e.g. pH equal to 1.
- aqueous suspensions comprising NCs according to the present invention may comprise ascorbic acid.
- said ascorbic acid is comprised in a %weight amount in the range from 0.1% to 0.5%, even more preferably from 0.1 to 0.3%, e.g., 0.2%.
- NCs according to the present invention are prepared using an amount in mg/ml of surfactant ranging from 20 mg/ml to 5 mg/ml, even more preferably ranging from 15 mg/ml to 7 mg/ml, e.g. an amount of 10 mg/ml.
- NCs according to the present invention are prepared using an amount in mg/ml of active substance of plant origin ranging from 20 mg/ml to 5 mg/ml, even more preferably ranging from 15 mg/ml to 7 mg/ml, e.g. an amount of 10 mg/ml.
- NCs according to the present invention are prepared using a weight ratio of surfactant (b) to active substance in the range form 1 :2 to 1:1.
- surfactant (b) to active substance in the range form 1 :2 to 1:1.
- the present invention also relates to compositions comprising said solid lipid nanoparticles or said nanocrystals and at least one pharmaceutically acceptable additive.
- compositions of the invention may be a pharmaceutical composition (or Live Biotherapeutic Products), a composition for a medical device, a dietary supplement, a food (or a novel food or a food for special medical purposes), a composition for a dietary supplement or food, or, alternatively, a composition for cosmetic use.
- Nanosystems according to the present invention can thus be advantageously used in various types of preparations (liquids, creams, sprays, gels, and foams) and for different applications (topical, oral, intravenous, intranasal, pulmonary, and ocular). Nanosystems according to the present invention are particularly advantageous for topical use.
- compositions according to the present invention are compositions formulated for use in medical devices.
- MDs medical devices
- EU European Parliament
- compositions that are particularly useful for use in Substance-based Medical Devices are described.
- Substance-based DMs can have therapeutic action.
- the actions that substance-based DMs can boast include, for example, mechanical, physical, chemical-physical, osmotic actions or more generally actions that take place at the extra-cellular level, such as barrier, lubricating, protective, and sequestering activities; this makes it a therapeutic device with a high safety profile.
- compositions comprising nanosystems according to the present invention can be advantageously used in substance-based DM.
- the Applicant has advantageously found that the nanosystems described in the present context can be used to prepare compositions comprising rosmarinic acid in a medical device for nasal use, effectively carrying that substance topically.
- composition comprising:
- composition comprising:
- SNs, NCs The nanosystems (SLNs, NCs) described in the present context, and compositions thereof are for medical use.
- nanosystems as described in the present invention can be advantageously used to topically carry active substances of plant origin, particularly to carry rosmarinic acid.
- nanosystems comprising rosmarinic acid can have antioxidant, anti-inflammatory, antiallergic, antimicrobial, and anti Quorum Sensing properties and can be advantageously administered in topical applications, preferably in nasal topical applications.
- nanosystems comprising RA and compositions thereof act on inflammatory processes.
- it has been observed how they interact with the proinflammatory protein HMGB1.
- It is an object of the present invention a method for alleviating/preventing allergies and/or inflammatory conditions in a subject comprising administering to the subject an effective amount of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same as described herein.
- the present disclosure further relates to the use of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, as described herein for alleviating/preventing allergies and/or inflammatory conditions.
- the present disclosure further relates to the use of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, as described herein for the manufacture of a substance-based medical device for alleviating/preventing allergies and/or inflammatory conditions.
- the present disclosure further relates to solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, for use in a method of treatment of allergies and/or inflammatory conditions.
- said nanosystems comprising RA and compositions comprising the same are for use in the treatment of inflammatory diseases affecting the nose including allergic rhinoconjunctivitis, and rhinosinusitis.
- allergic rhinoconjunctivitis means an inflammatory process of the nasal mucosa due to an immunologic reaction induced by, usually inhaled, allergens.
- compositions comprising nanosystems comprising RA are formulated in a nasal spray form and are for use in the treatment of inflammatory conditions of the nose.
- the present invention relates to a method for preventing or treating a disease or disorder associated with low-density lipoprotein oxidation, or the presence of reactive oxygen species (ROS), reactive nitrogen species and peroxynitrites, by administering nanosystems comprising RA and compositions comprising the same.
- ROS reactive oxygen species
- Said disease or disorder related to lipoprotein oxidation or the presence of reactive oxygen species can be selected from the group consisting of or alternatively comprising atherosclerosis, cancer and neurodegenerative diseases, Alzheimer's disease, and amyotrophic lateral sclerosis.
- Nanosystems comprising RA and compositions comprising said nanosystems are for use as antioxidants, and help to enhance the defense mechanism against reactive oxygen species (ROS), reactive nitrogen species and peroxynitrites.
- ROS reactive oxygen species
- the present invention relates to the use of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same as described herein, as antimicrobial agents.
- the present disclosure also refers to the use of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, as described herein for the manufacture of a substance-based medical device for use in an antimicrobial/antibacterial treatment.
- the present disclosure also refers to solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, for use in an antimicrobial/antibacterial method of treatment.
- said nanosystems comprising RA and compositions comprising the same are for use in a method of treatment against bacterial infections caused by Gram-positive and Gram-negative bacteria selected from the group consisting of or alternatively comprising Bacillus cereus, Salmonella infantis, Campylobacter jejuni and E. Coli, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA).
- Gram-positive and Gram-negative bacteria selected from the group consisting of or alternatively comprising Bacillus cereus, Salmonella infantis, Campylobacter jejuni and E. Coli, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA).
- the nanosystems described in the present context comprising rosmarinic acid can be administered as an adjuvant to a therapy with antibiotics selected from the group consisting of or alternatively comprising amoxicillin, ofloxacin, and vancomycin.
- the Applicant surprisingly found that the nanosystems described in the present context comprising rosmarinic acid can be advantageously used to reduce bacterial biofilm production during the early stages of formation; this is possible due to the interaction of RA with the cells "communication system", known as Quorum Sensing (QS).
- QS Quorum Sensing
- the anti-Quorum Sensing (QS) activity of the nanosystems comprising RA is particularly effective against bacteria such as Aeromonas Hydrophila and Pseudomonas Aeruginosa.
- Rosmarinic Acid (RA), batch BCCF0185, commercially available, purchased from Sigma-Aldrich, having a purity of 98%.
- Glyceryl monostearate purchased from A.C.E.F. Spa.
- NGM Topcithin purchased from A.C.E.F. Spa, (pharmaceutical/food grade soy lecithin).
- Nonionic surfactant Polysorbate 80 (IUPAC name: Polyoxyethylene (20) sorbitan monooleate) also known as Tween 80, purchased from A.C.E.F. Spa.
- D-o-Tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) (alpha-tocopherol, > 25%), purchased from A.C.E.F. Spa.
- Mucin solution 2mg/ml commercially available for purchase, (type II mucin, extracted from pig stomach).
- Simulated nasal fluid prepared using: type II mucin extracted from pig stomach, NaCI, KCI, CaCh ⁇ 2H 2 O, NaOH purchased from A.C.E.F. Spa.
- the instruments used in the present context are instruments commonly used in research laboratories, whose operation, measurement limits, sensitivities and accuracy are known to the person skilled in the art.
- Determination of particle size was done by Dynamic Light Scattering (DLS) measurements.
- the instrument used for light scattering measurements consists of:
- a sample housing cell consisting of a test tube with a capacity of 2-3 milliliters (cuvette), placed in the appropriate compartment and maintained at the desired temperature by a thermostatting system; (in this case, measurements are made at room temperature);
- Each datum reported refers to an average with standard deviation of three measurements representative of the average particle size.
- the curve related to particle size distribution is obtained by a parameter called polydispersion index (PDI).
- PDI polydispersion index
- the PDI provides direct information on the mono-dispersion (when the value is less than 0.4) or polydispersion (if the value is greater than 0.4) of the sample under examination.
- microelectrophoresis an electrokinetic process in which substances containing ionizable groups (ions or macromolecules) migrate under the action of an electric field.
- the speed with which the particles move is called “electrophoretic mobility” and is proportional to the size of the charge.
- electrokinetic mobility The speed with which the particles move.
- Measurements of potential C were made at 25°C. Through these size and potential C, measurements, not only the chemical and physical characteristics of the samples but also any phenomena related to stability over time can be studied.
- molecules in which the transition dipole is approximately parallel to the direction of light polarization are preferentially excited.
- fluorescence light is anisotropic: measuring in parallel polarized fluorescence (Z
- fluorescence anisotropy a quantity, called fluorescence anisotropy, defined as: is introduced.
- Anisotropy has a value of 1 for fully polarized light and 0 for isotropic light.
- r has a value, called fundamental anisotropy, that is determined solely by the type of fluorophore used: where a is the angle between the absorption dipole and the emission dipole of the fluorescent probe; ro can have values between 0.4 (parallel transition dipoles) and 0.2 (orthogonal dipoles).
- NEs-P, NEs P-RA, NEs-P RA-Chit, SLn, SLn-RA samples were prepared and then analyzed by spectrophotometer, using the following parameters:
- SLn and SLn-RA samples were diluted 1 :4 with Hepes buffer in order to avoid multiscattering phenomena.
- Glyceryl monostearate Glyceryl monostearate
- NGM-T NGM Topcithin
- Tween-80 (representing surfactant (b)) is used for the aqueous phase. (1% volume concentration).
- rosmarinic acid is added to the lipophilic phase previously solubilized in acetone, (in a 10mg/ml ratio).
- Table 1 shows the quali-quantitative composition of SLNs expressed in mg/ml.
- “SLN” indicates the sample without rosmarinic acid
- “SLN-RA” indicates the sample containing rosmarinic acid.
- the components of both phases were appropriately weighed with an analytical scale and, through the use of a heating plate, were brought to a temperature of 90 °C. RA was then solubilized in acetone, and the resulting solution was added to the lipophilic phase. The latter was then poured inside previously heated glass syringes.
- the lipophilic phase was quickly added to the solution of water and Tw-80 under turbine emulsifier and left to stir for 5 minutes at 9,000rpm.
- the resulting dispersion was then cooled with an ice bath under a turbine stirrer for 10 minutes until homogeneous solid particles having an average size of 230 nm, as measured by DLS measurements, were obtained.
- NCs were prepared by weighing 25g of demineralized water acidified with a 20% w/V hydrochloric acid solution to pH 1 (antisolvent) in which vitamin E TPGS (see materials) at 1% w/v was hot added (70 °C). Separately, 250 mg RA were solubilized in 0.5 ml acetone in order to obtain a final solution of 10 mg/ml. Acetone was selected as it can be easily removed by evaporation.
- Table 2 Composition of NCs expressed in mg/ml.
- the precipitate was obtained, it was centrifuged for 10 min at 4000 rpm. Subsequently, the supernatant was removed and the precipitate was redispersed at room temperature in 25 g of demineralized water under magnetic stirring.
- Selected SLNs and NCs formulations were characterized in terms of sizing, potential and PDI.
- Stability studies allow evaluation of the stability of nanosystems over time in terms of sizing, potential , entrapment efficiency and pH.
- DLS was used for sizing and potential measurements, UV spectrophotometry was used for entrapment/encapsulation efficiency, and electrode pH meter was used for pH. All samples are found to be stable over time and no significant changes are observed.
- the SLN sample (without active) maintained the initial parameters of hydrodynamic diameter and potential over time at both storage temperatures, showing no evident changes throughout the observation period.
- Table 5 shows the trend of RA concentration within the SLN-RA sample:
- RA is also found to be protected from degradative phenomena when prepared in nanocrystal form, as shown below in Table 6.
- Evaluation of the stability of the nanosystems according to the present invention also included observation of the pH values trend over time.
- the pH values of all test samples remained stable over time, in the accepted pH range for nasal administration, at both storage temperatures.
- the 70:30 ratio EtOH:FA solution was then selected as the diluent solution and the blank.
- UV analyses for RA-containing samples were performed in the absorbance range of 0-2.
- the calibration line was constructed, using EtOH:FA ratio 70:30 as the reference standard, this line relates the absorbance values, obtained by measuring the characteristic peak of RA, which is between 328-330 nm and the concentration of RA.
- Table 7 Dilutions used with respective concentrations and absorbances to construct the calibration line.
- Entrapment efficiency is expressed as the percentage of lipophilic active substance/probe entrapped within the nanosystem, with respect to the initial amount used in the formulation. Entrapment efficiency is a measure of the system's ability to entrap RA.
- mucin the main component of mucus
- SLN-RA sample The interaction between mucin (the main component of mucus) and the SLN-RA sample was studied by evaluating the differences in size and surface charge before and after the addition of the mucin suspension. The results are shown below in Table 9:
- NCs + M sample has a slightly increased hydrodynamic diameter compared to the NCs sample, while the surface charge remains almost unchanged.
- the increase in size suggests the presence of interactions with mucin that do not cause particle breakage.
- the release of the active substance by nanosystems is influenced by a number of parameters:
- the surface area involved in the passage the greater the area, the greater the amount of active substance that can be released.
- Membrane thickness is another factor influencing release; the thinner the membrane, the more rapid the passage.
- the hydroalcoholic solution was then withdrawn hourly and UV measured to monitor RA leakage through the membrane.
- Figure 11 shows the results obtained by analyzing the two samples in contact with SNF. From Figure 11, the different release profiles of the two formulations can be observed; in particular, during the first 5 minutes, the SLN-RA sample releases about 15% RA while NCs about 25%. Thereafter, RA release gradually increases in both formulations; in particular, a release of 60% in the first 30 minutes for NCs and 45% for SLNs is observed. After one hour, NCs release 90% RA, while SLNs release 100% after two hours.
- the experiment was carried out by placing the sample inside a "spray pump," which represents the device designed for the proposed application; after spraying the sample directly inside a cuvette, it was analyzed by DLS.
- Table 11 shows and compares data from nebulization tests dy spray dispenser of the two systems (NCs and SLNs); the changes in size and potential of the samples before and after nebulization (post-N) are then highlighted:
- NGM-T NGM Topicithin (soy lecithin)
- Results obtained from microbiological analyses carried out to verify the antibacterial activity of RA in alcoholic solution (N1) and on FP (N2) are shown in Tables 18 and 19.
- Table 18 shows the analysis for sample N1, in which time 0 of inoculation and analysis after 7 days are indicated.
- Table 19 shows a logarithmic reduction in the number of microorganisms as a function of time. The results show that sample N2 also shows a reduction after 7 days of contact of both inoculations (as shown in Figure 15).
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Abstract
The present invention relates to novel nanosystems optimized for the encapsulation and carrying of active molecules/substances of plant origin and preparation methods thereof. The present invention also relates to the use of these systems for the topical treatment of allergies and/or inflammation, preferably for the treatment of allergic rhinoconjunctivitis.
Description
NANOSYSTEMS FOR CARRYING/ENCAPSULATING INSOLUBLE MOLECULES OF PLANT ORIGIN, INCLUDING ROSMARINIC ACID
TECHNICAL FIELD
The present invention relates to novel nanosystems optimized for the encapsulation and carrying of active molecules/substances of plant origin and preparation methods thereof. The present invention also relates to the use of these systems for the topical treatment of allergies and/or inflammation especially affecting the nose.
STATE OF THE ART
Natural compounds, mainly of plant origin, have been the mainstay of traditional medicine for many years because of their many properties. To date, scientific research confirms that numerous natural substances can be a valuable support in the treatment or prevention of numerous diseases and/or disorders. For example, polyphenols have been shown to play a key role in the prevention and treatment of diseases. Rosmarinic acid (RA), in this regard, represents a substance of great biological interest, particularly promising in the medical field.
3-(3,4-Dihydroxyphenyl)-2R-{[3-(3,4-dihydroxyphenyl)prop-2E-enoyl]oxy}propanoic acid, more commonly known as Rosmarinic Acid (RA), is considered one of the most potent naturally occurring polyphenols. First isolated in 1958 from Rosmarinus officinalis, RA is contained in several plants belonging to the Lamiaceae family such as Perilla frutescens, which is its richest source, but it can also be found in other commonly used plants such as Ocimum basilicum, Origanum vulgare, Salvia officinalis, Melissa officinalis and many others.
One of the problems with rosmarinic acid and numerous other natural molecules/substances, especially of plant origin, is that they are poorly soluble in aqueous systems and tend to degrade over time due to their antioxidant nature, and very often they are difficult to formulate and administer for various therapeutic uses.
For this reason, there is a need to find new systems that allow active ingredients of natural origin to be easily formulated, making them usable in aqueous systems, and that allow for systems with high local or systemic availability that are easy to administer and nontoxic to the body.
There is also a felt need to find new encapsulation and carrying systems that make it possible to obtain safer and more effective therapeutic treatments through the carrying of natural active substances, with the aim of improving their biodistribution and toxicity profile as well as overcoming the chemical and physical limitations associated with the substances themselves.
The Applicant, following intensive and prolonged research and development activity, in order to solve the above technical problems has developed nanosystems capable of effectively encapsulating and carrying natural substances. Specifically, the Applicant has developed nanoparticles capable of encapsulating a variety of natural substances by making them usable in water-based systems and easy to administer. In addition, the Applicant has identified compositions comprising said nanosystems that are advantageously formulated for topical administration, preferably for topical nasal administration.
The Applicant surprisingly found that nanosystems prepared in the present context can be used for topical treatment of inflammatory diseases/disorders.
The nanosystems of the present invention and compositions thereof have no major side effects and can be administered to all categories of subjects, including pregnant or lactating women, or subjects with other comorbidities.
Finally, the nanosystems of the present invention and the compositions comprising them are easy to prepare and cost-effective.
These purposes and others, which will become clear from the detailed description that follows, are achieved by the nanosystems and compositions of the present invention through the technical features claimed in the appended claims.
SUMMARY OF THE INVENTION
The present invention relates to a solid lipid nanoparticle (NP) comprising or alternatively consisting of:
(a) a core comprising (I) a solid-state lipid phase and (II) rosmarinic acid; and
(b) at least one NP envelope comprising a surfactant.
The present invention also relates to a method for preparing said nanoparticle according to the present invention, wherein said method comprises the steps of:
(I) Hot addition of the lipophilic phase containing the active, to an aqueous phase, to obtain a two-phase system;
(II) Mixing said hot system obtained in step (I) with a turbine emulsifier until a homogeneous dispersion is obtained, preferably mixing at a speed from 6000 rpm to 10000 rpm, for example at 9000 rpm;
(II) Cooling under stirring the system obtained in step (II) to obtain solid particles with a lipid matrix, dispersed in an aqueous phase.
The present invention also relates to a nanocrystal comprising or alternatively consisting of:
(a) rosmarinic acid,
(b) at least one surfactant, where said surfactant is D-o-Tocopheryl polyethylene glycol 1000 succinate.
In addition, the present invention relates to a composition comprising the solid nanoparticle or the nanocrystal according to the present invention, and optionally at least one excipient of pharmacologically acceptable grade.
In addition, the present invention relates to said composition for use in a method of treatment of allergies and/or inflammatory conditions, preferably affecting the nose, or for antimicrobial/antibacterial use.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 : Preparative example scheme of Solid Lipid nanoparticles (SLNs).
Figure 2: Preparative example scheme of Nanocrystals (NCs).
Figures 3-8: Stability tests of the nanosystems object of the present invention. Figure 3: Stability at 25 °C of SLNs without active. Figure 4: Stability at 4 °C of SLNs without active. Figure 5: Stability at 25 °C of SLNs comprising rosmarinic acid. Figure 6: Stability at 4 °C of SLNs comprising rosmarinic acid.
Figure 7: Stability at 25 °C of the NCs.
Figure 8: Stability at 4 °C of the NCs.
Figure 9: Interaction times between the SLN-RA sample and mucin.
Figure 10: Interaction times between the NCs sample and mucin.
Figure 11 : Release profiles of SLNs with SNF and NCs with SNF.
Figures 12A and 12B: Stability studies on FP comprising SLN-RA conducted for a period of 15 days at 25 °C and 4 °C, respectively.
Figure 13A and 13 B: Stability studies on FP comprising NCs conducted for a period of 15 days at 25 °C and 4 °C, respectively.
Figure 14: Trend over time of the concentration of survived microorganisms in sample N1.
Figure 15: Trend over time of the concentration of survived microorganisms in sample N2.
Figure 16: Optical density profile showing the ability of RA to bind to HMGB-1.
Figure 17: Binding sites of HMGB-1 to rosmarinic acid.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to innovative nanosystems optimized for encapsulation, carrying and release of natural substances of plant origin, preferably for rosmarinic acid.
Specifically, the nanosystems described in the present context are advantageously usable in the medical, pharmaceutical, cosmetic, and nutraceutical fields, and allow safe and effective therapeutic treatments to be obtained through the carrying of natural substances, preferably rosmarinic acid, by overcoming the chemical and physical limitations associated with the substances themselves, while also improving their release profile.
In addition, the nanosystems described in the present context have high stability and allow localized release of the encapsulated active, reducing side effects and being particularly advantageous for topical use.
Through the use of the nanosystems according to the present invention, as described in detail below, carrying of lipophilic and/or highly sensitive natural substances is made possible, enabling stable systems capable of improving therapeutic efficiency.
Another advantage of the systems described in the present context relates to the reduction of side effects. The nanosystems, and compositions comprising them, according to the present invention, advantageously enable efficient carrying of natural active substances.
It is an object of the present invention a nanoparticle, said nanoparticle comprising at least one surfactant and a substance of plant origin, preferably rosmarinic acid, said substance of plant origin being encapsulated in said surfactant.
Preferably, said nanoparticle also comprises a lipid phase that encapsulates/encloses said substance of plant origin.
In a preferred embodiment, said nanoparticle according to the present invention is in the form either of solid lipid nanoparticle (SLN) or nanocrystals (NCs) comprising at least one active substance of plant origin, preferably comprising rosmarinic acid.
Solid lipid nanoparticles (SLNs) and nanocrystals according to the present invention will be described in detail below. For brevity these may also be referred to as "nanosystems”.
NANOSYSTEMS
SOLID LIPID NANOPARTICLE (SLN)
Advantageously, it is an object of the present invention a solid lipid nanoparticle comprising or alternatively consisting of:
(a) a core comprising or alternatively consisting of (i) a solid-state lipid phase and (ii) at least one active substance of natural origin selected from the group comprising or alternatively consisting of: curcumin, gingerols, rosmarinic acid and mixtures thereof;
(b) at least one surfactant.
Preferably, SLNs according to the present invention have sizes in the range from 10 nm to 1000 nm, even more preferably in the range from 20 nm to 600 nm, e.g., in the range from 50 nm to 200 nm, as measured by dynamic light scattering, DLS, measurements.
Preferably, SLNs according to the present invention have a weight ratio of lipid component to active substance in the range from 3:1 to 1 :1, e.g. a weight ratio of 2:1.
The at least one surfactant (b) used for the preparation of SLNs according to the present invention can be a non-toxic surfactant polymer.
Said surfactant may be a nonionic surfactant, for example, it may be a polysorbate selected from the group consisting of: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80 and mixtures thereof.
Preferably said surfactant is polysorbate 80.
In an embodiment, the at least one surfactant (b) is a phospholipid selected from the group comprising or alternatively consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and mixtures thereof.
In the present context, the term phospholipid refers to a lipid whose molecule has a hydrophilic "head" containing a phosphate group, and two hydrophobic "tails" derived from fatty acids, joined by a glycerol molecule.
Preferably said phospholipid used in the present context may be a glycerophospholipid, e.g., it may be selected from the group comprising or alternatively consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lecithin, phosphatidic acid, and mixtures thereof.
Preferably, a lecithin such as soy lecithin is used in the present context.
The at least one surfactant (b) advantageously surrounds and stabilizes the lipid core (a) comprising the active substance of plant origin.
Any wax (mixture) which is solid at the storage temperature of SLNs, i.e. at room temperature (about 25 °C), can be used for the lipid phase. For example, triglycerides, partial glycerides, fatty acids, steroids and/or waxes and lecithins can be used.
In a preferred embodiment, the lipid phase (i) of SLNs comprises or alternatively consists of Glyceryl monostearate (GMS) and a soy lecithin, e.g., NGM Topcithin (NMG-T) can be used as soy lecithin.
Surprisingly, when the lipid phase comprises Glyceryl monostearate (GMS) and/or a soy lecithin (e.g. NGM Topcithin (NGM-T)), better encapsulation/entrapment capabilities of the active substance are achieved.
The NGM Topcithin preferentially used in the present context is a phospholipid belonging to the lecithin family (soy lecithin). The use of this component in the lipid phase provides an additional stabilizing effect in addition to that of the surfactant (b).
SLNs described in the present context can be prepared using different preparation methods known to the person skilled in the art.
For example, SLNs can be prepared by high-pressure homogenization, hot/cold homogenization, emulsification and solvent evaporation.
Preferably, an easy-to-execute method that can be executed on a large scale is used in the present context.
Said SLNs preparation method comprises the steps of:
(I) Hot addition of the lipophilic phase containing the active, to an aqueous phase, to obtain a two-phase system;
(II) Mixing said hot system obtained in step (I) with a turbine emulsifier until a homogeneous dispersion is obtained, preferably mixing at a speed from 6000 rpm to 10000 rpm, for example at 9000 rpm;
(III) Cooling under stirring, e.g. under a turbine emulsifier, the system obtained in step (II) to obtain solid particles with a lipid matrix, dispersed in an aqueous phase.
Said preparation method of SLNs may also comprise a step of:
(la) Mixing the solubilized active substance in an organic solvent with the hot lipid phase. For example, if the active substance is rosmarinic acid, rosmarinic acid can be solubilized in acetone in a step (la). Since acetone is a volatile solvent, it is easily removed from the mixture obtained in step (la).
The lipid phase obtained in step (la) can be used in step (I) of hot addition and dispersion of said lipid phase with an aqueous phase.
Preferably step (I) is performed at a temperature from 60 °C to 95 °C, preferably at a temperature from 75 °C to 99 °C, e.g. at 90 °C.
Preferably, step (I) is performed using sterile disposable syringes. The lipid phase comprising the active substance of plant origin, e.g., rosmarinic acid, is heated and inserted into syringes that are then used for the hot addition of the lipid phase to the aqueous phase (step I).
SLNs according to the present invention have several advantages, including the ability to carry and entrap a high amount of active substance of natural origin, said active substance being preferably rosmarinic acid. SLNs according to the present invention are stable systems that are well tolerated by the body and do not pose toxicity risks.
Advantageously, the SLNs described in the present context allow for a controlled and localized release of the active substance, particularly rosmarinic acid.
It was also observed that SLNs according to the present invention advantageously enable improved targeting of rosmarinic acid. Finally, SLNs according to the present invention have high stability and bioavailability.
A preferred embodiment refers to a solid lipid nanoparticle comprising (a) a core comprising (i) a solid- state lipid phase and (ii) rosmarinic acid,
(b) at least one surfactant.
A preferred embodiment refers to a solid lipid nanoparticle comprising (a) a core comprising (i) a solid- state lipid phase and (ii) rosmarinic acid,
(b) at least one surfactant, wherein said lipid phase comprises a mixture of Glycerylmonostearate (GMS) and NGM Topcithin (NGM- T), and wherein said surfactant (b) is a polysorbate, preferably it is polysorbate 80.
The SLNs described in the present context can be advantageously dispersed in an aqueous phase. Preferably, the aqueous phase may comprise surfactant (b).
The pH of the aqueous dispersion comprising SLNs can be adjusted by adding a base, or an acid, in order to obtain the suitable pH for the desired application.
In a preferred embodiment, the pH is adjusted in the range from 3.5 to 6.8. This pH is particularly advantageous in applications for nasal use. Samples have shown high stability in this pH range.
Preferably, SLNs according to the present invention are prepared using an amount in mg/ml of lipid phase ranging from 30 mg/ml to 5 mg/ml, even more preferably ranging from 25 mg/ml to 10 mg/ml, e.g. an amount of 18 mg/ml.
Preferably, SLNs according to the present invention are prepared using an amount in mg/ml of surfactant ranging from 20 mg/ml to 5 mg/ml, even more preferably ranging from 15 mg/ml to 7 mg/ml, e.g. an amount of 10 mg/ml.
Preferably, SLNs according to the present invention are prepared using an amount in mg/ml of active substance of plant origin ranging from 20 mg/ml to 5 mg/ml, even more preferably ranging from 15 mg/ml to 7 mg/ml, e.g. an amount of 10 mg/ml.
NANOCRYSTALS (NCs)
In the present context the term "nanocrystals" (NCs) refers to "carrier-free" colloidal drug delivery systems. In an embodiment, active substances of plant origin, preferably rosmarinic acid, are synthesized in the form of nanocrystals stabilized by surfactants or surfactant polymers.
It is an object of the present invention a nanocrystal comprising or alternatively consisting of (a) an active substance of plant origin and (b) at least one surfactant.
The present invention also relates to nanocrystals stabilized by at least one surfactant or surfactant polymer stably dispersed in an aqueous system.
It is therefore an object of the present invention an aqueous dispersion comprising nanocrystals comprising or alternatively consisting of (a) an active substance of plant origin, e.g., rosmarinic acid, and (b) at least one surfactant.
Preferably, it is an object of the present invention an aqueous dispersion comprising nanocrystals comprising or alternatively consisting of:
(a) rosmarinic acid,
(b) at least one surfactant.
Advantageously, rosmarinic acid NCs according to the present invention allow effective carrying of the active substance, the structure of the nanocrystal being entirely composed of that active substance. They thus enable a sufficiently high therapeutic concentration to be achieved for the desired effect.
Preferably, NCs, in a nano-suspension form, have an average size in the range from 10 nm to 800 nm, even more preferably in the range from 10 nm to 500 nm, e.g, in the range from 10 to 100 nm, as measured by dynamic light scattering, DLS, measurements.
NCs according to the present invention can occur in crystalline, semi-crystalline or amorphous form.
The nanocrystal NCs described in the present context can be synthesized using techniques known to the person skilled in the art, such as top-down or bottom-up synthesis techniques.
The former, considered high-energy methods, such as dry/wet milling or high-pressure homogenization, involve particle diameter reduction, starting from a large size crystal.
The latter, on the other hand, involve low-energy methods, such as spray drying or nanoprecipitation, based on the aggregation of crystalline or amorphous particles.
A preferred synthesis method in the present context is nanoprecipitation, which consist of the rapid addition of a solvent (in which the active substance is solubilized), to the antisolvent, resulting in the formation of a precipitate of active.
The at least one surfactant (b) used to stabilize NCs according to the present invention can be selected from the group comprising or alternatively consisting of: polysorbate 20, polysorbate 80, a polaxamer, and mixtures thereof. As a polaxamer can be used, for example, Lutrol F68, which is commercially avilable.
Preferably, the at least one surfactant (b) is D-o-Tocopheryl polyethylene glycol 1000 succinate, also known as vitamin E TPGS.
Advantageously, the use of vitamin E TPGS to stabilize NCs according to the present invention enables NCs of rosmarinic acid to be obtained with high stability. This substance, formed by esterification of vitamin E succinate with polyethylene glycol 1000, has been extensively studied for its emulsifying, dispersing, gelling and solubilizing effects on poorly water-soluble substances.
Vitamin E TPGS is a derivative of vitamin E, which has antioxidant properties similar to those of tocopherol. It has been used in the past as an excipient to overcome multidrug resistance (MDR) and has been approved by the U.S. Food and Drug Administration (FDA) as a pharmaceutically safe adjuvant.
Advantageously, NCs according to the present invention make it possible to increase the dissolution rate of the active substance of plant origin, preferably rosmarinic acid, ameliorating issues related to low bioavailability and reduced permeation of the latter.
In addition, NCs according to the present invention are considered low-risk and readily biodegradable, as well as widely adhesive to membranes/mucous membranes. Finally, these systems allow the use of low concentrations of excipients thus reducing excipient-related toxic effects.
The nanocrystals NCs according to the present invention can be prepared by the following method comprising the steps of:
(i) Preparing an aqueous solution (antisolvent) with acidic pH, preferably with a pH from 4 to 1, hot adding surfactant (b), preferably by adding 1% vitamin E TPGS;
(ii) Separately solubilizing the active substance in a volatile organic solvent, e.g., solubilizing rosmarinic acid in acetone until a solution having a concentration preferably ranging from 3 mg/ml to 20 mg/ml is obtained, e.g., 10 mg/ml;
(iii) Adding the solution prepared in step (ii) to the antisolvent solution prepared in step (i), magnetically stirring the sample during addition;
(iv) Heating the sample in step (iii) to help the evaporation of the organic solvent and obtaining the NCs by precipitation.
NCs can be redispersed in demineralized water, and the pH can be adjusted according to application needs.
Step (i) of the NCs preparation method according to the present invention can be carried out at a temperature from 90 °C to 50 °C, preferably from 80 °C to 65 °C, e.g. at 70 °C.
Step (iv) of the NCs preparation method according to the present invention can be carried out at a temperature from 90 °C to 50 °C, preferably from 75 °C to 55 °C, e.g. at 60 °C.
Step (i) of preparing an acidified aqueous solution can be carried out by using a hydrochloric acid solution e.g. acidification by adding a 20% wA/ hydrochloric acid solution until the desired pH is obtained, e.g. pH equal to 1.
Advantageously, aqueous suspensions comprising NCs according to the present invention may comprise ascorbic acid. Preferably said ascorbic acid is comprised in a %weight amount in the range from 0.1% to 0.5%, even more preferably from 0.1 to 0.3%, e.g., 0.2%.
Preferably, NCs according to the present invention are prepared using an amount in mg/ml of surfactant ranging from 20 mg/ml to 5 mg/ml, even more preferably ranging from 15 mg/ml to 7 mg/ml, e.g. an amount of 10 mg/ml.
Preferably, NCs according to the present invention are prepared using an amount in mg/ml of active substance of plant origin ranging from 20 mg/ml to 5 mg/ml, even more preferably ranging from 15 mg/ml to 7 mg/ml, e.g. an amount of 10 mg/ml.
Preferably, NCs according to the present invention are prepared using a weight ratio of surfactant (b) to active substance in the range form 1 :2 to 1:1.
COMPOSITIONS WITH FOCUS ON NASAL DEVICE
The present invention also relates to compositions comprising said solid lipid nanoparticles or said nanocrystals and at least one pharmaceutically acceptable additive.
Said compositions of the invention, according to any one of the described embodiments, may be a pharmaceutical composition (or Live Biotherapeutic Products), a composition for a medical device, a dietary supplement, a food (or a novel food or a food for special medical purposes), a composition for a dietary supplement or food, or, alternatively, a composition for cosmetic use.
Nanosystems according to the present invention can thus be advantageously used in various types of preparations (liquids, creams, sprays, gels, and foams) and for different applications (topical, oral, intravenous, intranasal, pulmonary, and ocular). Nanosystems according to the present invention are particularly advantageous for topical use.
Preferably, the compositions according to the present invention are compositions formulated for use in medical devices.
In the present context, medical devices (MDs) refer to instruments, equipments and products, intended for use on humans for therapeutic purposes. These are regulated by Regulation (EU) 2017/745 of the European Parliament (MDR745), which replaced the old Directive 93/42.
In the present context, compositions that are particularly useful for use in Substance-based Medical Devices are described.
Substance-based DMs, as defined in the regulation, can have therapeutic action. The actions that substance-based DMs can boast include, for example, mechanical, physical, chemical-physical, osmotic actions or more generally actions that take place at the extra-cellular level, such as barrier, lubricating, protective, and sequestering activities; this makes it a therapeutic device with a high safety profile.
Compositions comprising nanosystems according to the present invention can be advantageously used in substance-based DM.
The Applicant has advantageously found that the nanosystems described in the present context can be used to prepare compositions comprising rosmarinic acid in a medical device for nasal use, effectively carrying that substance topically.
It is an object of the present invention a composition comprising:
- a solid lipid particle or nanocrystal, as described in the present context, and optionally at least one additive of pharmaceutically acceptable grade.
In particular, it is an object of the present invention a composition comprising:
- a solid lipid particle or nanocrystal, as described in the present context;
- at least one sodium hyaluronate (SH) as a mucoadhesive and viscosifying agent,
- potassium sorbate (PS),
- sodium benzoate (SB) as preservatives,
- sodium chloride (SC) to obtain an isosmotic formulation, wherein said composition is formulated as a nasal spray.
MEDICAL USES
The nanosystems (SLNs, NCs) described in the present context, and compositions thereof are for medical use.
The Applicant has advantageously found that nanosystems as described in the present invention can be advantageously used to topically carry active substances of plant origin, particularly to carry rosmarinic acid.
The Applicant has advantageously found that nanosystems comprising rosmarinic acid, and compositions comprising said nanosystems, can have antioxidant, anti-inflammatory, antiallergic, antimicrobial, and anti Quorum Sensing properties and can be advantageously administered in topical applications, preferably in nasal topical applications.
Advantageously, nanosystems comprising RA and compositions thereof act on inflammatory processes. In particular, it has been observed how they interact with the proinflammatory protein HMGB1.
It is an object of the present invention a method for alleviating/preventing allergies and/or inflammatory conditions in a subject comprising administering to the subject an effective amount of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same as described herein. The present disclosure further relates to the use of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, as described herein for alleviating/preventing allergies and/or inflammatory conditions. The present disclosure further relates to the use of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, as described herein for the manufacture of a substance-based medical device for alleviating/preventing allergies and/or inflammatory conditions. The present disclosure further relates to solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, for use in a method of treatment of allergies and/or inflammatory conditions.
Preferably, said nanosystems comprising RA and compositions comprising the same are for use in the treatment of inflammatory diseases affecting the nose including allergic rhinoconjunctivitis, and rhinosinusitis.
In the present context, allergic rhinoconjunctivitis means an inflammatory process of the nasal mucosa due to an immunologic reaction induced by, usually inhaled, allergens.
Advantageously, compositions comprising nanosystems comprising RA are formulated in a nasal spray form and are for use in the treatment of inflammatory conditions of the nose.
In another aspect, the present invention relates to a method for preventing or treating a disease or disorder associated with low-density lipoprotein oxidation, or the presence of reactive oxygen species (ROS), reactive nitrogen species and peroxynitrites, by administering nanosystems comprising RA and compositions comprising the same.
Said disease or disorder related to lipoprotein oxidation or the presence of reactive oxygen species (ROS) can be selected from the group consisting of or alternatively comprising atherosclerosis, cancer and neurodegenerative diseases, Alzheimer's disease, and amyotrophic lateral sclerosis.
Nanosystems comprising RA and compositions comprising said nanosystems are for use as antioxidants, and help to enhance the defense mechanism against reactive oxygen species (ROS), reactive nitrogen species and peroxynitrites.
In another aspect, the present invention relates to the use of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same as described herein, as antimicrobial agents.
The present disclosure also refers to the use of solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, as described herein for the manufacture of a substance-based medical device for use in an antimicrobial/antibacterial treatment.
The present disclosure also refers to solid lipid nanoparticles or nanocrystals comprising rosmarinic acid, or compositions comprising the same, for use in an antimicrobial/antibacterial method of treatment.
Preferably, said nanosystems comprising RA and compositions comprising the same are for use in a method of treatment against bacterial infections caused by Gram-positive and Gram-negative bacteria selected from the group consisting of or alternatively comprising Bacillus cereus, Salmonella infantis, Campylobacter jejuni and E. Coli, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA).
Advantageously, the nanosystems described in the present context comprising rosmarinic acid can be administered as an adjuvant to a therapy with antibiotics selected from the group consisting of or alternatively comprising amoxicillin, ofloxacin, and vancomycin.
In addition, the Applicant surprisingly found that the nanosystems described in the present context comprising rosmarinic acid can be advantageously used to reduce bacterial biofilm production during the early stages of formation; this is possible due to the interaction of RA with the cells "communication system", known as Quorum Sensing (QS).
The anti-Quorum Sensing (QS) activity of the nanosystems comprising RA, described herein, is particularly effective against bacteria such as Aeromonas Hydrophila and Pseudomonas Aeruginosa.
EXPERIMENTAL PART
MATERIALS
The following is the list of materials and instruments used in preparing illustrative examples of some embodiments of the present invention.
Rosmarinic Acid (RA), batch BCCF0185, commercially available, purchased from Sigma-Aldrich, having a purity of 98%.
Glyceryl monostearate, purchased from A.C.E.F. Spa.
NGM Topcithin purchased from A.C.E.F. Spa, (pharmaceutical/food grade soy lecithin).
Nonionic surfactant Polysorbate 80 (IUPAC name: Polyoxyethylene (20) sorbitan monooleate) also known as Tween 80, purchased from A.C.E.F. Spa.
D-o-Tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) (alpha-tocopherol, > 25%), purchased from A.C.E.F. Spa.
Regenerated cellulose membrane MWCO 8kDa from Spectra/Por®.
Mucin solution 2mg/ml, commercially available for purchase, (type II mucin, extracted from pig stomach).
Simulated nasal fluid (SNF) prepared using: type II mucin extracted from pig stomach, NaCI, KCI, CaCh ■ 2H2O, NaOH purchased from A.C.E.F. Spa.
A solution of mucin extracted from pig stomach 2 mg/ml in Hepes buffer was used for mucoadhesion studies.
The instruments used in the present context, are instruments commonly used in research laboratories, whose operation, measurement limits, sensitivities and accuracy are known to the person skilled in the art.
INSTRUMENTS
- Precision Scale: Sartorius
- PHmeter (Phenomenal): VWR
- Magnetic stirrer with heating plate: AREC.X
- Turbine emulsifier (Silverson L5M)
- Dynamic Light Scattering (Malvern ZetaSizer Nano ZS 90) (A equal to 6770 A (red laser))
- Perkin-Elmer Lamba 25 UV Spectrophotometer
- HPLC (Chromaster): VWR
- Centrifuge (Rotina380): Hettich
- Sonicator
- Ubbelohde Viscometer
- Osmometer (Osmomat 300): Gonatech
METHODS
SIZING WITH DYNAMIC LIGHT SCATTERING
Determination of particle size was done by Dynamic Light Scattering (DLS) measurements.
Schematically, the instrument used for light scattering measurements consists of:
A light source represented by a laser capable of generating a 10 mW light beam with A equal to 6770 A (red laser);
A sample housing cell consisting of a test tube with a capacity of 2-3 milliliters (cuvette), placed in the appropriate compartment and maintained at the desired temperature by a thermostatting system; (in this case, measurements are made at room temperature);
A detector, placed at a fixed angle 9 = 90°, consisting of a digital autocorrelator, capable of calculating, as a result of collecting and recording intensities at successive times, the products and averages of the discrete summation defining the correlation function C (q, T); A correlator that enables the calculation of the correlation function; A data collection and processing system, which enables the correlation functions to be obtained.
Measurements with DLS were made on all prepared samples (SLn, NC) by diluting the obtained suspensions 1:100 (v:v) with Hepes buffer) in order to avoid multiscattering phenomena.
Measurements were conducted at 25°C and performed for characterization, stability and mucoadhesion studies of the prepared nanosystems, as described in the present context.
Each datum reported refers to an average with standard deviation of three measurements representative of the average particle size.
The curve related to particle size distribution is obtained by a parameter called polydispersion index (PDI).
The PDI provides direct information on the mono-dispersion (when the value is less than 0.4) or polydispersion (if the value is greater than 0.4) of the sample under examination.
DETERMINATION OF SURFACE CHARGE (POTENTIAL )
With DLS it is also possible to calculate the potential C, of particles, a parameter that defines their associated charge.
To measure potential , the instrument uses microelectrophoresis, an electrokinetic process in which substances containing ionizable groups (ions or macromolecules) migrate under the action of an electric field. The speed with which the particles move is called "electrophoretic mobility" and is proportional to the size of the charge.
To measure the potential , the sample is placed inside a special microelectrophoresis cell, at the ends of which an electric field of known value is applied, having standard values typically used in this type of measurement.
Measurements of potential C, were made at 25°C. Through these size and potential C, measurements, not only the chemical and physical characteristics of the samples but also any phenomena related to stability over time can be studied.
FLUORESCENCE ANISOTROPY
This technique makes it possible to determine the diffusive motions of fluorescent molecules in solution and in membranes. In fluorescence anisotropy measurements, the sample is excited with linearly polarized light to realize a photo-selection phenomenon.
In this regard, molecules in which the transition dipole is approximately parallel to the direction of light polarization are preferentially excited.
In the absence of diffusive motion, on the other hand, fluorescence light is anisotropic: measuring in parallel polarized fluorescence (Z||) and perpendicularly polarized fluorescence (/±) with respect to the direction of excitation, a higher intensity of the former is observed with respect to the latter.
To quantify this phenomenon, a quantity, called fluorescence anisotropy, defined as:
is introduced.
Anisotropy has a value of 1 for fully polarized light and 0 for isotropic light. In the case where the motions are negligible (as is the case with an extremely viscous and/or low-temperature solution), r has a value, called fundamental anisotropy, that is determined solely by the type of fluorophore used:
where a is the angle between the absorption dipole and the emission dipole of the fluorescent probe; ro can have values between 0.4 (parallel transition dipoles) and 0.2 (orthogonal dipoles).
If the sample is excited with a pulse of light and the trend of anisotropy over time is monitored, a decay of r from the initial r0 value to 0 (when the orientation has become totally random) is observed.
This decay is all the more rapid the more rapid the motions of the molecule. It is well known that an increase in fluorescence anisotropy should be traced to a loss of membrane fluidity. In SLN samples, the membrane is the lipophilic compartment within which the fluorescent probe is solubilized in both NEs and SLNs.
Fluidity/rigidity measurements were conducted on samples containing the probe 1,6-diphenyl-1,3,5- hexatriene (DPH), a strongly lipophilic molecule that fits into the hydrophobic internal phase.
To measure fluorescence anisotropy, a 2- 10-4 M solution of DPH was prepared by solubilizing the latter in chloroform. From the resulting solution, 440 p. I was taken and evaporated for 24 h to remove the chloroform.
In the same tube in which DPH in a solid form was obtained, NEs-P, NEs P-RA, NEs-P RA-Chit, SLn, SLn-RA samples (prepared as given below in the text) were prepared and then analyzed by spectrophotometer, using the following parameters:
Aex =400nm
Aem =425nm slit 5/5
SLn and SLn-RA samples were diluted 1 :4 with Hepes buffer in order to avoid multiscattering phenomena.
PREPARATION OF SOLID LIPID NANOPARTICLES (SLN)
For the preparation of SLNs without rosmarinic acid (used as a reference), two solid at room temperature waxes are used for the lipophilic phase: Glyceryl monostearate (GMS) and NGM Topcithin (NGM-T) (see materials).
A solution of Tween-80 (Tw-80) (representing surfactant (b)) is used for the aqueous phase. (1% volume concentration).
For the preparation of SLNs with rosmarinic acid, the same procedure is used, but rosmarinic acid is added to the lipophilic phase previously solubilized in acetone, (in a 10mg/ml ratio).
The amounts used are shown in Table 1 :
Table 1
Table 1 shows the quali-quantitative composition of SLNs expressed in mg/ml. "SLN" indicates the sample without rosmarinic acid, while "SLN-RA," indicates the sample containing rosmarinic acid.
For sample preparation, the components of both phases (lipid and aqueous) were appropriately weighed with an analytical scale and, through the use of a heating plate, were brought to a temperature of 90 °C. RA was then solubilized in acetone, and the resulting solution was added to the lipophilic phase. The latter was then poured inside previously heated glass syringes.
Once the suitable temperature (90°C) was reached, the lipophilic phase was quickly added to the solution of water and Tw-80 under turbine emulsifier and left to stir for 5 minutes at 9,000rpm.
The resulting dispersion was then cooled with an ice bath under a turbine stirrer for 10 minutes until homogeneous solid particles having an average size of 230 nm, as measured by DLS measurements, were obtained.
To achieve a pH of 6, 10 pil of 10% w/v NaOH solution were added.
PREPARATION OF NANOCRISTAL (NCs)
NCs were prepared by weighing 25g of demineralized water acidified with a 20% w/V hydrochloric acid solution to pH 1 (antisolvent) in which vitamin E TPGS (see materials) at 1% w/v was hot added (70 °C). Separately, 250 mg RA were solubilized in 0.5 ml acetone in order to obtain a final solution of 10 mg/ml. Acetone was selected as it can be easily removed by evaporation.
The amounts of the components used are shown in Table 2:
Table 2: Composition of NCs expressed in mg/ml.
The solution of RA in acetone was added rapidly to the antisolvent by Gilson pipette and placed under magnetic stirring at 600rpm for 10 minutes. To promote the evaporation of acetone and thus the precipitation of RA, the sample was heated at 60°C for two hours.
Once the precipitate was obtained, it was centrifuged for 10 min at 4000 rpm. Subsequently, the supernatant was removed and the precipitate was redispersed at room temperature in 25 g of demineralized water under magnetic stirring.
To the resulting clear and pale yellow solution, a drop of 20% wA/ NaOH solution was finally added by Pasteur pipette to reach the desired pH and 0.2% ascorbic acid (w/v).
Surprisingly, the addition of ascorbic acid to the sample, maintains the clear color of the sample (it slows degradative processes).
CHEMICAL AND PHYSICAL CHARACTERIZATION
HYDRODYNAMIC DIAMETER AND POTENTIAL <
Selected SLNs and NCs formulations were characterized in terms of sizing, potential and PDI.
Table 3
It can be seen from the data shown in Table 3 that, for SLNs, there is a slight increase in particle size following the introduction of RA. The latter, in addition to affecting the size of the nanosystems, also influences their potential values, which tend to become more negative; this change in surface charge suggests the arrangement of RA on the surface of the nanosystem (SLN).
STABILITY TESTS
Stability studies allow evaluation of the stability of nanosystems over time in terms of sizing, potential , entrapment efficiency and pH.
All samples tested for stability over time were stored at two different temperatures: 25°C and 4°C away from light, for a period of 60 days.
For all samples, the following were then monitored over time: hydrodynamic diameter, potential , EE (entrapment efficiency) and pH, as shown in Figures 3-8.
Stability studies over time, carried out in order to show any changes in size and potential , were conducted on SLNs and NCs; For SLN samples, studies were performed on both SLNs (without active) and RA-containing samples (SLN-RA, NCs). All parameters mentioned above were analyzed daily during the first seven days and weekly for the next three months.
DLS was used for sizing and potential measurements, UV spectrophotometry was used for entrapment/encapsulation efficiency, and electrode pH meter was used for pH.
All samples are found to be stable over time and no significant changes are observed.
In detail, as shown in Figures 3 and 4, the SLN sample (without active) maintained the initial parameters of hydrodynamic diameter and potential over time at both storage temperatures, showing no evident changes throughout the observation period.
The same results are obtained for the SLN-Ra samples (comprising rosmarinic acid). The results are shown in Figure 5 and Figure 6. Stability studies of the SLN-RA sample showed a nearly constant trend over time in the values of hydrodynamic diameter and potential at both 25 °C and 4 °C without significant changes. Therefore, it can be said that the sample remains stable over time and that the addition of RA does not affect the structure of the system.
Stability studies of the NCs sample showed a constant trend over time in the values of hydrodynamic diameter and potential at both temperatures (results shown in Figure 7 and Figure 8). It is therefore possible to say that the sample remains stable over time.
In addition to conducting stability in terms of hydrodynamic diameter and potential , the variation of RA in hydroalcoholic solution (70:30 EtOH:FA) and within SLN-RA and NCs samples, both at 25 °C and 4 °C, was analyzed over time. Since RA has a strong tendency to oxidation, its concentration was monitored over time in order to check for possible degradative phenomena and to hypothesize possible protection of RA by the nanosystem structure. Table 4 shows the variation over time of RA concentration, monitored for 30 days, in hydroalcoholic solution.
Table 4
From Table 4, shown above, it is possible to observe a decrease in RA concentration, probably attributable to the degradation phenomenon, in the aliquot placed at room temperature, starting from day 15. In contrast, in that placed at 4°, the concentration of RA showed no decrease throughout the observation period. Thus, it can be stated that, RA at room temperature tends to degrade over time and remain stable at the temperature of 4°C when dissolved in hydroalcoholic solution.
Table 5 shows the trend of RA concentration within the SLN-RA sample:
Table 5
From Table 5, shown above, it can be observed that RA, when carried in SLN and stored at 4 °C, remains stable over time from day 1 to day 30, while there is a nonsignificant change in the aliquot placed at 25 °C. The data show that the SLN system appears to be able to better protect RA at room temperature from the degradative phenomena it undergoes when solubilized in hydroalcoholic solution and not carried.
RA is also found to be protected from degradative phenomena when prepared in nanocrystal form, as shown below in Table 6.
Table 6
Evaluation of the stability of the nanosystems according to the present invention also included observation of the pH values trend over time. The pH values of all test samples remained stable over time, in the accepted pH range for nasal administration, at both storage temperatures.
QUANTITATIVE ANALYSIS OF ROSMARINIC ACID
Evaluation of entrapment/encapsulation efficiency.
Quantitative analysis of rosmarinic acid by UV spectrophotometry.
To perform UV analysis of the samples, the solubility of RA in hydroalcoholic solutions in different ratios of Ethanol (EtOH):Hepes buffer was initially evaluated.
The 70:30 ratio EtOH:FA solution was then selected as the diluent solution and the blank.
All samples (SLNs and Nanocrystals) were analyzed. The samples were appropriately diluted (at a ratio of 1 :50 for the NCs and SLN-RA samples) and then analyzed using a dual-beam UV-Vis spectrophotometer, which allows simultaneous readout of the sample and the reference standard by absorbance measurements of incident light over a range of wavelengths operating in both the ultraviolet and visible regions.
UV analyses for RA-containing samples were performed in the absorbance range of 0-2.
For greater accuracy in determining the concentration of RA within the nanosystems, the calibration line was constructed, using EtOH:FA ratio 70:30 as the reference standard, this line relates the absorbance values, obtained by measuring the characteristic peak of RA, which is between 328-330 nm and the concentration of RA.
Initially, RA was solubilized in the standard solution and then serial dilutions were made until different concentrations (expressed in mg/ml) were obtained for different absorbance values (Abs), as shown in Table 7:
Table 7: Dilutions used with respective concentrations and absorbances to construct the calibration line.
Since the R2 factor of the line is greater than 0.99, it can be said that this line follows a linear trend. Thanks to the calibration line, it was possible to evaluate the entrapment efficiency, i.e. the concentration of RA (expressed in mg/ml) entrapped within the nanosystem.
Entrapment efficiency (EE) is expressed as the percentage of lipophilic active substance/probe entrapped within the nanosystem, with respect to the initial amount used in the formulation. Entrapment efficiency is a measure of the system's ability to entrap RA.
EE is calculated by the following formula:
In addition to EE determination, pH was appropriately measured in order to keep the samples in the proposed range for nasal application (3.5-6.8).
Table 8 below shows the EE and pH values obtained:
Table 8
Regarding the EE-related analyses, it was found that both systems have very good entrapment efficiency, i.e. they are able to trap a high amount of RA.
MUCOADHESION STUDIES
For mucoadhesion studies, 2 mg/ ml of mucins were suspended in Hepes buffer (pH 6.1) and the resulting suspension was stirred overnight at 34 °C.
Specific parameters including temperature, concentration and pH value were appropriately selected and monitored in order to simulate the same conditions present in the nasal cavity.
The interaction between mucin (the main component of mucus) and the SLN-RA sample was studied by evaluating the differences in size and surface charge before and after the addition of the mucin suspension. The results are shown below in Table 9:
Table 9
From Table 9, it can be seen that the SLN-RA + M (Mucin) sample, although it does not show much change in terms of potential does show an increased hydrodynamic diameter compared to the SLN-RA sample. This increase suggests the presence of interactions with mucin that do not cause particle breakage.
Particle size and potential C, were measured by DLS at time 0, 5, 10 and 15 minutes in order to determine the time required for the formation of the mucin-nanocrystal (NCs) complex and mucin SLN complex. The results show that once the complex is formed the size remains stable and is as shown in Table 9.
Furthermore, as shown in Figure 9, it can be observed that the interaction between the sample and mucin is instantaneous and the increased size of SLN-RA + M is maintained over the three-hour period. Mucoadhesive studies were also conducted on the NCs sample as shown below in Table 10 and Figure 10.
Table 10
From the results it can be seen that the NCs + M sample has a slightly increased hydrodynamic diameter compared to the NCs sample, while the surface charge remains almost unchanged. The increase in size suggests the presence of interactions with mucin that do not cause particle breakage.
As shown in Figure 10, the interaction between the sample and mucin is instantaneous, and the increased size of NCs + M is maintained over the three-hour period.
To support mucoadhesion studies, samples were contacted with simulated nasal fluid (SNF) maintaining a 1 :1 ratio, these studies confirm the results obtained with mucin.
RELEASE STUDIES
The release of the active substance by nanosystems is influenced by a number of parameters:
First of all, the surface area involved in the passage: the greater the area, the greater the amount of active substance that can be released.
Membrane thickness is another factor influencing release; the thinner the membrane, the more rapid the passage.
Finally, the difference in concentration of the active at the two sides of the membrane: the higher the difference in concentration at the two sides, he more rapid the passage.
In order to evaluate the amount of RA released in the unit of time, different release studies were conducted using a mucin solution and a solution of SNF (simulated nasal fluid).
Releases were conducted by adding 550 pil of sample and 450 pil of either mucin or SNF solution inside a regenerated cellulose membrane, properly closed to form a "candy." The latter was then immersed in a beaker containing EtOH:FA 70:30 as the external phase, under magnetic stirring.
The hydroalcoholic solution was then withdrawn hourly and UV measured to monitor RA leakage through the membrane.
These release studies, carried out in both mucin and SNF contact, were conducted in order to determine and compare the ability of the nanosystems (NCs and SLNs) to release RA over 3 hours.
Figure 11 shows the results obtained by analyzing the two samples in contact with SNF. From Figure 11, the different release profiles of the two formulations can be observed; in particular, during the first 5 minutes, the SLN-RA sample releases about 15% RA while NCs about 25%. Thereafter, RA release gradually increases in both formulations; in particular, a release of 60% in the first 30 minutes for NCs and 45% for SLNs is observed. After one hour, NCs release 90% RA, while SLNs release 100% after two hours.
NEBULIZATION STUDIES
These studies, conducted on SLN-RA and NCs samples, were aimed at verifying that the physicalchemical characteristics of the samples following spraying remain unchanged.
The experiment was carried out by placing the sample inside a "spray pump," which represents the device designed for the proposed application; after spraying the sample directly inside a cuvette, it was analyzed by DLS.
Table 11 shows and compares data from nebulization tests dy spray dispenser of the two systems (NCs and SLNs); the changes in size and potential of the samples before and after nebulization (post-N) are then highlighted:
Table 11
What emerges is that both nanosystems (SLNs and NCs) are suitable for delivery by nasal spray because they showed no significant changes in hydrodynamic diameter and surface charge.
FINISHED PRODUCT FORMULATION comprising SOLID LIPID NANOPARTICLES
GMS: Glyceryl monostearate
NGM-T: NGM Topicithin (soy lecithin)
RA: rosmarinic acid
SH: sodium hyaluronate
PS: potassium sorbate
SB: sodium benzoate
SC: sodium chloride v-TPGS: vitamin E TPGS
Table 12
FINISHED PRODUCT FORMULATION comprising NANOCRYSTALS.
Table 13
After the preparation, the following parameters were measured:
• pH, by electrode pHmeter: pH:4.7
• density, by pycnometer: 1 g/cm3
• viscosity, by Ubbelohde viscometer: 3.06
• osmolality, by osmometer: 300
Specifically, for FP (finished product) the following were analyzed: size, potential , pH, and EE (%) to verify that these characteristics did not vary from the SLN-RA and NCs samples upon addition of other substances.
Other crucial parameters were then monitored, including density, viscosity and osmolality. The FP data are shown in Table 14 below.
Table 14
From the results shown in Table 14, it can be observed that the characteristics of size, potential and EE (%) remain unchanged compared with the SLN-RA and NCs samples; in particular, the moderate increase in particle diameter found in FP can be easily attributed to the presence of SH.
Regarding the pH data, it can be observed that the addition of the various components within the formulation does not significantly affect the pH, which remains in the range suitable for the proposed
application (nasal). Density, viscosity and osmolality were also evacuate for FP, parameters that are suitable for the administration of a product for nasal use.
STABILITY STUDIES OF THE FINISHED PRODUCT
Stability studies conducted on FP for a period of 15 days showed the results shown in Figure 12 A and B and Figure 13 A and B.
From Figures 12 (A and B) and 13 (A and B), it can be observed that in the time interval in which it was monitored, PF showed excellent stability in terms of both size and potential change at both storage temperatures.
Also for a period of 15 days, the stability of RA within the FP was monitored as shown in Table 15 and Table 16:
Table 15
Table 16
NEBULIZATION STUDIES ON THE FINISHED PRODUCT (FP)
Nebulization studies were also conducted on FP in order to verify that the final formulation was suitable for nebulization by spray dispenser. For this purpose, the size and surface charge were analyzed before and after the nebulization experiment; Table 17 shows the data obtained:
Table 17
From the results shown in Table 17, it can be seen that the FP prepared using the two nanosystems (SLNs and NCs) is found to be suitable for nebulization by spray dispenser, as the characteristics of size and potential remain unchanged from the starting samples.
MICROBIOLOGICAL ANALYSIS ON THE FINISHED PRODUCT
Results obtained from microbiological analyses carried out to verify the antibacterial activity of RA in alcoholic solution (N1) and on FP (N2) are shown in Tables 18 and 19. Table 18 shows the analysis for sample N1, in which time 0 of inoculation and analysis after 7 days are indicated.
Table 18
From the data shown in Table 18, a logarithmic reduction in the number of microorganisms as a function of time can be observed (NR means "no recovery").
The results of the analysis show that sample N1 shows a reduction after 7 days of contact of both inoculations (Figure 14).
The same analysis conducted on sample N2, showed the following results reported in Table 19 and Figure 15:
Table 19
Table 19 shows a logarithmic reduction in the number of microorganisms as a function of time. The results show that sample N2 also shows a reduction after 7 days of contact of both inoculations (as shown in Figure 15).
In conclusion, it can be stated that both samples show excellent antibacterial activity against the tested strains and that this activity in FP does not vary from that of RA in alcohol solution.
PHARMACOLOGICAL STUDIES
The optical density trend of the various samples analyzed (NCs and SLNs), as a function of RA concentration, was elaborated (Figure 16).
From the data obtained, it can be observed that RA has a strong interaction with HMGB-1; this interaction is confirmed by further molecular docking studies, through which it is possible to observe the binding of RA with this protein at two specific binding sites, one at higher energy (-6.5 Kcal/mol) and the other at lower energy (-7.9 Kcal/mol) (Figure 17).
From Figure 17, it can be observed that while the highest energy binding site is not highly populated, i.e. few RA conformations are located in this binding pocket, the lower energy binding site represents the most suitable binding site for RA.
In conclusion, it can be said that RA interacts with HMGB-1 protein with higher affinity for the lower-energy pocket.
Claims
1 . A solid lipid nanoparticle (NP) comprising or alternatively consisting of:
(a) a core comprising (I) a solid-state lipid phase and (ii) rosmarinic acid;
(b) at least one NP envelope comprising a surfactant.
2. The nanoparticle according to claim 1, wherein said lipid phase is selected from the group comprising or alternatively consisting of Glycerylmonostearate (GMS), NGM Topcithin, and mixtures thereof.
3. The nanoparticle according to any one of claims 1-2, wherein said surfactant is a polysorbate, preferably it is polysorbate 80.
4. A method for preparing the nanoparticle according to any one of claims 1-3, wherein said method comprises the steps of:
Said SLN preparation method comprises the steps of:
( l ) Hot addition of the lipophilic phase containing the active, to an aqueous phase, to obtain a two- phase system;
( ii ) Mixing said hot system obtained in step (I) with a turbine emulsifier until a homogeneous dispersion is obtained, preferably mixing at a speed from 6000 rpm to 10000 rpm, for example at 9000 rpm;
( m ) Cooling under stirring the system obtained in step (II) to obtain solid particles with a lipid matrix, dispersed in an aqueous phase.
5. A nanocrystal comprising or alternatively consisting of:
(a) rosmarinic acid,
(b) at least one surfactant, wherein said surfactant is D-o-Tocopheryl polyethylene glycol 1000 succinate.
6. A composition comprising the solid nanoparticle according to any one of claims 1-3, or the nanocrystal according to claim 5, and optionally at least one excipient of pharmacologically acceptable grade.
7. The composition according to claim 6 for use as a medicament.
8. The composition for use according to claim 7, wherein said composition is for use in a method of treatment of allergies and/or inflammatory conditions.
9. The composition for use according to claim 8, wherein said use is for the treatment of allergic rhinoconjunctivitis.
10. The composition for use according to claim 7, wherein said use is for antimicrobial/antibacterial treatment.
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Non-Patent Citations (3)
| Title |
|---|
| ANA RAQUEL MADUREIRA ET AL: "Characterization of solid lipid nanoparticles produced with carnauba wax for rosmarinic acid oral delivery", RSC ADVANCES, vol. 5, no. 29, 1 January 2015 (2015-01-01), pages 22665 - 22673, XP055464439, DOI: 10.1039/C4RA15802D * |
| ATIKORN PANYA ET AL: "Interactions between α-Tocopherol and Rosmarinic Acid and Its Alkyl Esters in Emulsions: Synergistic, Additive, or Antagonistic Effect?", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 60, no. 41, 17 October 2012 (2012-10-17), pages 10320 - 10330, XP055165333, ISSN: 0021-8561, DOI: 10.1021/jf302673j * |
| CAMPOS DÉBORA A ET AL: "Optimization of the production of solid Witepsol nanoparticles loaded with rosmarinic acid", COLLOIDS AND SURFACES B: BIOINTERFACES, ELSEVIER AMSTERDAM, NL, vol. 115, 21 November 2013 (2013-11-21), pages 109 - 117, XP028836497, ISSN: 0927-7765, DOI: 10.1016/J.COLSURFB.2013.10.035 * |
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