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WO2022084929A1 - Nanoparticles for transport across the blood-brain barrier - Google Patents

Nanoparticles for transport across the blood-brain barrier Download PDF

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Publication number
WO2022084929A1
WO2022084929A1 PCT/IB2021/059750 IB2021059750W WO2022084929A1 WO 2022084929 A1 WO2022084929 A1 WO 2022084929A1 IB 2021059750 W IB2021059750 W IB 2021059750W WO 2022084929 A1 WO2022084929 A1 WO 2022084929A1
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Prior art keywords
acid
cholesterol
constituent
nanoparticles
ligand
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French (fr)
Inventor
Giulia BIROLINI
Elena Cattaneo
Flavio Forni
Pasquale LINCIANO
Barbara RUOZI
Giovanni TOSI
Marta VALENZA
Maria Angela Vandelli
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Universita degli Studi di Milano
Universita Degli Studi di Modena e Reggio Emilia
Istituto Nazionale di Genetica Molecolare INGM
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Universita degli Studi di Milano
Universita Degli Studi di Modena e Reggio Emilia
Istituto Nazionale di Genetica Molecolare INGM
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons

Definitions

  • the present invention relates to nanoparticles (NPs) for the delivery of active ingredients across the blood-brain barrier, in which said nanoparticles comprise at least one ligand bound on the surface of a colloidal system, characterized in that said at least one ligand is a fatty acid, and/or derivatives thereof, and said colloidal system comprises or consists of at least one constituent and, optionally, one or more active ingredients of interest.
  • NPs nanoparticles
  • the present invention further relates to the use of the same NPs to deliver active ingredients in the central nervous system.
  • the blood-brain barrier has the function of defending the central nervous system (CNS) from the intrusion of potentially toxic or otherwise exogenous microorganisms, particles, or molecules. Therefore, the BBB is an obstacle to the therapeutic approach of diseases affecting the CNS, such as brain tumors, stroke, ischemia, aging, and cognitive decline associated therewith, Alzheimer’s, Parkinson’s, Huntington’s, where the active ingredients hardly reach the action site in adequate concentration.
  • Solid lipid nanoparticles are extensively studied. Serpe L et al. in Europ. J. Pharm. Sci. 2010; 39: 428-436 describe SLNs comprising cholesteryl butyrate as a lipid matrix. Said SLNs are successfully used to deliver antiinflammatory drugs in the intestine.
  • EP401301 describes a prodrug consisting of a hexa-unsaturated fatty acid (docosahexaenoic acid, or cervonic acid), having 22 carbon atoms, to which dopamine is bound by amide bond.
  • the authors demonstrate the compound’s ability to overcome the BBB and, once in the CNS, hydrolyze to release the active ingredient at the desired action site.
  • SLNs having a diameter ranging between 70 and 200 nm, with a polydispersion index (PDI) between 0.2 and 0.65 and a Zeta potential of -28/29 mV.
  • PDI polydispersion index
  • Such SLNs comprise about 45% w/w of cholesterol conjugated with butyric acid.
  • the cholesterol included in these SLNs is exclusively cholesterol conjugated with butyric acid, free cholesterol not being loaded in said SLNs.
  • the present invention relates to nanoparticles comprising fatty acids and/or derivatives thereof bound on the surface of colloidal systems comprising one or more active ingredients of interest.
  • A, B Mice treated with col-BUT NPs.
  • C, D Mice treated with col NPs.
  • E, F Mice treated with Col-BUT-Glu NPs.
  • G, H Mice treated with Col-Phe-BUT NPs.
  • I, J Mice treated with BODIPY-BUT NPs.
  • STR striatum
  • CX cortex
  • mice 3 mice/NP type mice obtained 4 hours after intraperitoneal injection of 1.2 mg of the indicated NPs.
  • A, B Mice treated with PLGA-BUT NPs.
  • STR striatum
  • CTX cortex
  • mice 3 mice/NP type mice obtained 4 hours after intraperitoneal injection of 1.2 mg of the indicated NPs.
  • A, B Mice treated with col-LIN NPs.
  • C, D Mice treated with col NPs.
  • Figure 5 representative images of the organized matrix formed by the col- BUT NPs according to the present invention suspended in water (A) or of col- BUT conjugates suspended in water (B, comparison).
  • At least one means that at least one type of said component is included in the preparation, or in an embodiment the preparation comprises only one type of said component, in a further embodiment two types of said component are included.
  • at least one ligand indicates that the preparation comprises at least one type of ligand, i.e., in an embodiment the preparation comprises a ligand which is butyric acid, in a further embodiment it is to be understood that the preparation comprises two ligands, by way of example butyric acid and linoleic acid.
  • a nanoparticle comprising at least one fatty acid, or derivatives thereof, which is referred to herein as a ligand, bound on the surface of a colloidal system which comprises or consists of a constituent and, optionally, one or more active ingredients of interest.
  • Said fatty acid, or derivative is bound by a chemical bond to at least one functional group which is present on one or more of the constituents of said colloidal system, so as to form a constituent-ligand conjugate.
  • the carboxylic group of the fatty acid or the derivative thereof is conjugated by means of an ester bond with a hydroxyl group of the constituent, or by means of an amide bond with an amino functional group of the constituent.
  • said functional group of the constituent or of the ligand is activated, according to methods known to those skilled in the art, in order to facilitate the conjugation reaction.
  • the carboxyl group is optionally activated by means of an ester with an amide or a diamide to then form an amide with an amino functional group or a carboxyl group, respectively, or the carboxyl group is activated in the form of anhydride or by means of activating agents such as EDC (1-ethyl-3-(3- dimethylaminopropyljcarbodiimide) or DCC (dicyclohexylcarbodiimide).
  • the colloidal system is obtained according to NP preparation methods known to those skilled in the art.
  • the NPs according to the present invention comprise at least one conjugate and at least one constituent and, optionally, one or more further active ingredients.
  • said at least one conjugate is present in an amount between 3 and 25% w/w, or between 5 and 20% w/w, or between 8 and 15% w/w, preferably about 10% w/w and said at least one constituent and, optionally, one or more further active ingredients, is present in amounts between 75 and 97% w/w, or between 80 and 95% w/w, or between 85 and 92% w/w, preferably about 90% w/w.
  • Said conjugate consists of at least one ligand chemically bound to at least one constituent.
  • said at least one ligand is a fatty acid, preferably a linear or branched fatty acid, optionally substituted and/or esterified, saturated or unsaturated, with a number of carbon atoms between 4 and 22 or between 4 and 18. In an embodiment, it is a short-chain fatty acid, with a number of carbon atoms between 4 and 6, or medium-chain, with a number of carbon atoms between 6 and 12, or long-chain, comprising up to 22 carbon atoms, optionally substituted and/or esterified.
  • said fatty acid is a saturated or unsaturated linear fatty acid.
  • said fatty acid is selected from the group comprising: optionally substituted and/or esterified butyric acid, valeric acid, palmitic acid, margaric acid, stearic acid, arachic acid, palmitoleic acid, oleic acid, elaidinic acid, vaccenic acid, gadoleic acid, linoleic acid, a linoleic acid, stearidonic acid, arachidonic acid, thymnodonic acid, clupanodonic acid, cervonic acid.
  • said fatty acid is butyric acid.
  • said fatty acid is linoleic acid.
  • said fatty acid is substituted with phenyl, or it is esterified with glucose.
  • said fatty acid is 2-phenylbutyric acid (Phe-BUT).
  • said fatty acid is butyric acid esterified with glucose (Glu- BUT).
  • said constituent is any suitable substance for preparing NPs, in particular it is selected from the group comprising cholesterol and biodegradable and biocompatible polymers and/or copolymers, preferably poly(lactic-co-glycolic) acid (PLGA).
  • said constituent is cholesterol.
  • Said at least one ligand which is a fatty acid or derivative is bound to said constituent by chemical bond to at least one functional group of said constituent, forming said conjugate.
  • said conjugate comprises a ligand which is butyric acid, optionally substituted and/or esterified, or linoleic acid, optionally substituted and/or esterified, and a constituent which is cholesterol, or PLGA.
  • said conjugate is selected from the group comprising butyric acid - cholesterol (col-BUT), 2-phenylbutyric acid - cholesterol (col-Phe-BUT), butyric acid - Glucose - cholesterol (col-Glu-BUT), linoleic acid - cholesterol (col-LIN), butyric acid - PLGA (PLGA-BUT).
  • the NPs according to the present invention comprise a conjugate and a constituent, where said conjugate is present in an amount between 3 and 25% w/w, or between 5 and 20% w/w, or between 8 and 15% w/w, preferably about 10% w/w, and said constituent is present in an amount between 75 and 97% w/w, or between 80 and 95% w/w, or between 85 and 92% w/w, preferably about 90% w/w.
  • said NPs also comprise one or more further active ingredients selected among the active ingredients which have the target thereof in the CNS.
  • said one or more further active ingredients are selected among antibacterials, antivirals, psychotropic drugs, antidepressants, anticancer drugs, active ingredients for treating migraines, analgesics, antagonists, or agonists of receptors present in the CNS, antibodies, antisense oligonucleotides, hormones, diagnostic agents such as paramagnetic complexes for MRI, radionuclides for nuclear medicine or for diagnosis.
  • said active ingredient corresponds to said constituent.
  • said constituent which is also the only active ingredient is cholesterol.
  • the NP population according to the present invention has unique features because it is extremely homogeneous both in terms of size, which is between 230 and 350 nm, preferably between 245 and 330 nm, and of PDI (square of the standard deviation divided by the average particle diameter) which is between 0.15 and 0.22, preferably between 0.17-0.21.
  • the measured Z potential or electrokinetic potential (ZP) is between -10 and - 21 mV.
  • the present invention further relates to a method for preparing NPs comprising:
  • ligand which is at least one fatty acid or derivative
  • NPs preferably selected from the group comprising cholesterol or biodegradable and biocompatible polymers or copolymers, preferably poly(lactic-co-glycolic) acid (PLGA).
  • PLGA poly(lactic-co-glycolic) acid
  • said nanometric colloidal system is obtained through nanoprecipitation followed by evaporation of the solvent or alternatively by fusion and emulsion, resulting in the formation of NPs with a matrix shape and structure and homogeneous sizes in the order of a few hundred nanometers.
  • said conjugate and said constituent and, optionally said at least one further active ingredient are dissolved in organic solvents, at a temperature between room temperature and 70°C.
  • Said solution comprising said conjugate and said constituent and, optionally, said further active ingredient is slowly added to an aqueous solution comprising a surfactant, preferably Pluronic F68, where said Pluronic F68 is present in said aqueous solution in an amount between 1.5 - 5% w/V, or between 2 and 3% w/V, or about 2.5% w/V, keeping under stirring.
  • a surfactant preferably Pluronic F68
  • said solvent is preferably selected from the group comprising MeOH and EtOH and said nanoprecipitation occurs between 35 and 70°C.
  • said constituent is PLGA
  • said solvent is preferably acetone or acetonitrile and said nanoprecipitation occurs at room temperature.
  • the sample thus formulated is kept under stirring until complete evaporation of the solvent.
  • the present invention further relates to a NP according to the invention for use in the release of active ingredients in the CNS.
  • said use is for treating diseases related to an altered metabolism of cerebral cholesterol.
  • said use is for treating neurological, neurodegenerative diseases, selected for example from Parkinson's, Alzheimer’s, Huntington’s, aging, and cognitive decline associated therewith.
  • said use is for treating Huntington’s disease.
  • the NPs according to the present invention in the embodiment where said constituent is cholesterol and cholesterol is also the only active ingredient charged thereon, comprise an amount of cholesterol which is near 100% by weight of the NP itself.
  • the NPs according to the present invention effectively cross the blood-brain barrier, in addition to showing good stability features, are therefore capable of delivering a high amount of cholesterol within the CNS, overcoming the problems encountered with the systems currently available based on NPs of an insufficient amount of cholesterol capable of being delivered to the CNS.
  • Example 1 synthesis of the butyric acid-cholesterol conjugate
  • the butyric acid-cholesterol conjugate was synthesized by direct esterification of the hydroxyl group of cholesterol with butyric anhydride in the presence of triethylamine as a base (Diagram 1 ).
  • Cholesterol 500 mg, 1.30 mmol; 1 eq.
  • butyric anhydride (2,135 ml, 13.0 mmol, 10 eq.)
  • triethylamine 216 pl, 1.56 mmol, 1.2 eq.
  • the organic phase was washed in succession with a saturated solution of Na2COs, 2N HCI and finally with a saturated solution of NaCI.
  • the organic phase was dried on anhydrous Na2SC and concentrated.
  • the 2-phenylbutyric acid-cholesterol conjugate was synthesized by adapting the protocol used in example 1 as follows (diagram 2).
  • cholesterol (667 mg, 1.72 mmol, 1 eq.), 2-phenylbutyric anhydride (1 .0 mL, 3.45 mmol, 2 eq.) and triethylamine (286 pL, 2.06 mmol, 1 .2 eq.) were solubilized in anhydrous DCM inside a Schlenk tube at room temperature and under nitrogen atmosphere. The tube was sealed, and the mixture stirred at 50°C for 24 hours. After cooling to room temperature and dilution with ethyl acetate, the organic phase was washed in succession with a saturated solution of Na2COs, 2N HCI and a saturated solution of NaCI, then dried on anhydrous Na2SO4 and concentrated.
  • the identity and purity of the phenylbutyric acid-cholesterol conjugate were confirmed by 1 H-NMR and 13 C-NMR spectroscopy (Bruker 400 with 1 H at 400.134 MHz and 13 C at 100.62 MHz).
  • Cholesteryl linoleate (PL02) was synthesized by direct esterification of the cholesterol hydroxyl group with linoleic acid using EDC as activating agent for carboxylic acid and dimethylaminopyridine (DMAP) as catalyst, as shown in diagram 3:
  • EDC HCI (624 mg, 3.25 mmol, 2.5 equiv), DMAP (16 mg, 0.13 mmol, 0.1 equiv) and cholesterol (500 mg, 1.30 mmol, 1 equiv) were added to a solution of linoleic acid (728 l, 2.35 mmol, 1 .8 equiv) in anhydrous DCM at 0°C and under nitrogen atmosphere.
  • the reaction was stirred at room temperature for 18 hours.
  • the solvent was then removed, and the residue was dissolved in ethyl ether.
  • the organic phase was washed with a saturated solution of Na2COs, 10% HCI and brine.
  • the organic phase was dried on anhydrous Na2SO4 and concentrated.
  • the crude was purified on silica gel using hexane/AcOEt 9:1 to give 700 mg (yield 85%) of a white waxy solid.
  • the synthesis of the butyric acid - fatty acid derivatized cholesterol conjugate with a glucose was performed as shown in diagram 4. To allow the simultaneous bond of cholesterol and glucose to the butyric acid, this was substituted with a succinate which maintains the same size and properties as butyric acid but has two functional groups which are exploited to bind the constituent and glucose.
  • the cholesterol was reacted with succinic anhydride, in the presence of triethylamine as a base and a catalytic amount of DMAP in anhydrous toluene at 60°C overnight, to obtain the cholesterol hemisuccinate PLF09.
  • PLF07 trimethylsilyl ether
  • Diagram 4 Reagents and conditions: a) cholesterol (1 equiv.), succinic anhydride (1.6 equiv.), triethylamine (2 equiv.), DMAP (cat.), anhydrous toluene, 60°C, overnight, yield 87%; b) glucose (1 equiv.), TMSCI (6 equiv.), hexamethyldisilazane (6 equiv.), pyridine, 0°C at rt, 2 days, yield 93%; c) ammonium acetate (2 equiv.), DCM: MeOH 1 :1 v/v, rt, 6 h, quantitative yield; d) PLF09 (1 equiv.), PLF08 (1 equiv.), DCC (1.3 equiv.), DMAP (cat.), anhydrous THF, N 2 , 60°C, 2 hours, yield 62%; e) TFA (22 equiv.), DCM, 0°C, 1
  • the protective group on the second nitrogen atom was then removed by hydrogenolysis using sodium borohydride as a hydrogen source and catalyzed by 10% Pd/C.
  • the reaction was carried out in methanol at room temperature for 4 hours to provide the amine intermediate PLF14 with quantitative yield.
  • PLF15 Diagram 5 Reagents and conditions, a) N-Cbz-ethylenediamine (1 equiv), butyryl chloride (2 equiv), triethylamine (3 equiv), anhydrous DCM, N 2 , room temperature, 18 hours, quantitative yield; b) PLF13 (1 equiv), 10% Pd/C (0.1 equiv), NaBH4 (1 equiv), methanol, room temperature, 4 hours, quantitative yield; c) PLF14 (320 mg, 1 equiv), PLGA (320 mg), EDC HCI (1 equiv), HOBt (1 equiv), triethylamine (2 equiv), anhydrous DCM, room temperature, 18 hours, yield 65%.
  • Triethylamine (905 uL, 6.51 mmol, 3 equiv) and butyryl chloride (451 uL, 4.35 mmol, 2 equiv) are added to a suspension of N-Cbz-ethylenediamine hydrochloride (500 mg, 2.17 mmol, 1 equiv) in anhydrous DCM at room temperature and under nitrogen atmosphere. It is left to react under the same conditions for 18 hours, after which the solvent is evaporated. The residue is taken up with ethyl acetate and the organic phase is washed in succession with 10% HCI, a saturated solution of Na2COs and finally with a saturated solution of NaCI. The organic phase is dried on anhydrous Na2SO4 and concentrated. 560 mg (98% yield) of a white solid are obtained. The spectral characterization is consistent with the literature data.
  • EDC HCI (536 mg, 2.75 mmol, 1 equiv), HOBt (371 mg, 2.75 mmol, 1 equiv), PLF14 (320 mg, 2.75 mmol, 1 equiv) and triethylamine (763 uL, 5.50 mmol, 2 equiv.) are added to a solution of PLGA (320 mg) in anhydrous DCM at room temperature and under an inert atmosphere. It is left to react under the same conditions for 18 hours, after which the solvent is removed by filtration. The crude is triturated on diethyl ether and the precipitate discarded. The filtrate is concentrated and ground on methanol.
  • NP preparation col-BUT NPs comprising butyric acid as ligand and cholesterol as constituent and sole active ingredient were prepared as follows by nanoprecipitation.
  • the cholesterol-butyric acid conjugate prepared as in example 1 and cholesterol were provided.
  • a labeled cholesterol was also provided, i.e. , cholesterol-Cy5 (Invitrogen) or BODIPY cholesterol.
  • the components were dissolved in MeOH at 60°C. The following ratios were used: 10% w/w butyric acid-cholesterol conjugate, 85% w/w cholesterol, 5% w/w cholesterol-Cy5 (or cholesterol-Bodipy).
  • the hot solution was slowly added to an aqueous solution of Pluronic F68 (2.5% w/V) kept under stirring. Once the nanoparticles were formed, the stirring was continued until the organic solvent evaporated.
  • col-Phe-BUT NPs were obtained, where the conjugate is phenylbutyric acid (Phe-BUT) conjugated with cholesterol, col-Glu-BUT NPs, where the conjugate is glucose-modified butyric acid conjugated with cholesterol and col-LIN NPs where the conjugate is linoleic acid conjugated with cholesterol.
  • Said NPs are obtained with the same constituent which is cholesterol at the same concentrations and under the same experimental conditions described above for the col-BUT NPs.
  • PLGA-BUT NPs were obtained by nanoprecipitation starting from butyric acid as ligand and PLGA (PM about 11 ,000, lactic/glycolic ratio 50:50) as constituent.
  • the components were provided in the following % w/w ratios: 10% PLGA-BUT, 88% PLGA, 2% PLGA-Cy5.
  • the NPs were obtained after solubilization of the components in acetone and subsequent addition to an aqueous solution at room temperature of Pluronic F68 (2.5% w/v) and kept under stirring. Once the NPs formed, the stirring was continued until the organic solvent evaporated.
  • PCS photon correlation spectroscopy
  • NPs structure and morphology, which characterize them by defining the peculiar technological-pharmaceutical properties thereof.
  • a microscopic analysis was carried out with SEM-FEG transmission microscope.
  • col-BUT NPs obtained as in example 6 were suspended in MilliQ water and observed under transmission electron microscope.
  • the exemplary photographs in Figure 5A show that they form a matrix structure, organized in nanometer-sized particles, thus defining a new nanometric matrix system.
  • conjugates of example 1 and 2 were analyzed. As is clear from Figure 5B, said conjugates are not organized in a defined structure, they are configured as disorganized lipid chains.
  • Figure 2A shows the images obtained from striatum sections
  • Figure 2B from cortex sections of mice treated with col-BUT NPs
  • Figures 2C and 2D show a representative image of what was obtained with NPs consisting of cholesterol and dye, therefore without the ligand, in striatum and cortex, respectively. No passage of the blood-brain barrier is observed.
  • Figures 2A and 2B, according to the invention show the presence of NP signals inside the cerebral parenchyma, unequivocally signifying the passage across the BBB observed with col-BUT NPs. Similar results were shown both in the striatum and in the cortex for each type of tested NP according to the present invention.
  • col-BUT-Glu NPs Figure 2E striatum and 2F cortex
  • col-Phe-BUT NPs Figure 2G striatum and 2H cortex
  • BODIPY-BUT NPs BODIPY-BUT NPs
  • the PLGA-BUT NPs also demonstrated the ability to cross the BBB, by virtue of the conjugation of PLGA with the ligand BUT ( Figure 3A striatum and 3B cortex). In the absence of the ligand, the PLGA NPs do not reach the CNS, as widely described in the literature (Del Grosso A et al. Sci Adv. 2019; 5(11 ):eaax7462).
  • Figure 4 shows sections of striatum (Figure 4A) and cortex (Figure 4B) of mice treated with NPs obtained using cholesterol conjugated with the ligand linoleic acid (col-LIN NPs) and related controls without ligand ( Figure 4C and 4D).
  • the data described here show that the NPs according to the present invention effectively cross the BBB and are found in the cortex sections and in the striatum sections.

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Abstract

The present invention relates to nanoparticles for the delivery of active ingredients across the blood-brain barrier, wherein said nanoparticles comprise at least one ligand bound on the surface of a colloidal system, wherein said at least one ligand is at least one linear or branched fatty acid, optionally substituted and/or esterified, saturated or unsaturated, with a number of carbon atoms between 4 and 22 and said colloidal system comprises or consists of at least one constituent and, optionally, one or more active ingredients, characterized in that said ligand is bound by a chemical bond to at least one functional group which is present on one or more of the constituents of said colloidal system, so as to form a constituent-ligand conjugate.

Description

“Nanoparticles for transport across the blood-brain barrier”
The present invention relates to nanoparticles (NPs) for the delivery of active ingredients across the blood-brain barrier, in which said nanoparticles comprise at least one ligand bound on the surface of a colloidal system, characterized in that said at least one ligand is a fatty acid, and/or derivatives thereof, and said colloidal system comprises or consists of at least one constituent and, optionally, one or more active ingredients of interest.
The present invention further relates to the use of the same NPs to deliver active ingredients in the central nervous system.
Background art
The blood-brain barrier (BBB) has the function of defending the central nervous system (CNS) from the intrusion of potentially toxic or otherwise exogenous microorganisms, particles, or molecules. Therefore, the BBB is an obstacle to the therapeutic approach of diseases affecting the CNS, such as brain tumors, stroke, ischemia, aging, and cognitive decline associated therewith, Alzheimer’s, Parkinson’s, Huntington’s, where the active ingredients hardly reach the action site in adequate concentration.
For this reason, an increasing amount of research has focused on the identification of non-invasive means capable of allowing active ingredients of pharmacological interest to cross the BBB and reach the CNS. Numerous studies have focused on directing drugs to the CNS using colloidal systems (liposomes, nanoparticles, etc.). Such systems can transport active substances adsorbed or covalently bound on the surface thereof, or incorporated therein (Xie J et al. Biomaterials. 2019; 224: 119491 ).
Solid lipid nanoparticles (SLNs) are extensively studied. Serpe L et al. in Europ. J. Pharm. Sci. 2010; 39: 428-436 describe SLNs comprising cholesteryl butyrate as a lipid matrix. Said SLNs are successfully used to deliver antiinflammatory drugs in the intestine.
Gonzalez-Paredes A et al. in Europ. J. Pharmac. and Biopharmac. 2019; 134: 166-177 describe SLNs as a system for delivering oligonucleotides with antimicrobial effect inside bacterial cells.
EP401301 describes a prodrug consisting of a hexa-unsaturated fatty acid (docosahexaenoic acid, or cervonic acid), having 22 carbon atoms, to which dopamine is bound by amide bond. The authors demonstrate the compound’s ability to overcome the BBB and, once in the CNS, hydrolyze to release the active ingredient at the desired action site.
Recent studies have shown that cholesterol, delivered to the CNS by polymeric NPs, led to a recovery of cognitive decline but not of motor defect and other neuropathological parameters in a mouse model of Huntington’s disease (Valenza M et al. EMBO Mol Med 2015; 7: 1547-1564). The recovery observed was partial because the amount of cholesterol delivered by said polymeric NPs proved to be sub-optimal. In order to obtain the desired therapeutic effect, it is necessary to inject larger quantities or proceed with closer administrations over time.
In order to overcome this limit, increasing doses of cholesterol were provided centrally in the same animal model with the aid of osmotic mini-pumps (Birolini et al. EMBO Mol Med 2020; e12519). The effects observed on the phenotype were positive, demonstrating that cholesterol is a valid agent in the therapeutic treatment of Huntington’s disease. However, systems based on mini-pumps are invasive systems, not applicable to the treatment of chronic patients.
Brioschi A et al. in Molecules 2008; 13: 230-254 describe SLNs having a diameter ranging between 70 and 200 nm, with a polydispersion index (PDI) between 0.2 and 0.65 and a Zeta potential of -28/29 mV. Such SLNs comprise about 45% w/w of cholesterol conjugated with butyric acid. The cholesterol included in these SLNs is exclusively cholesterol conjugated with butyric acid, free cholesterol not being loaded in said SLNs.
Therefore, the need remains strongly felt to have efficient systems to facilitate the BBB crossing of active ingredients the action site of which is in the CNS.
Description of the invention
The present invention relates to nanoparticles comprising fatty acids and/or derivatives thereof bound on the surface of colloidal systems comprising one or more active ingredients of interest.
Description of the drawings Figure 1 : 1H-NMR spectrum of the cholesterol-butyric acid conjugate with peak integration at 4.58-4.66 ppm (H-3) and 2.25 (H-2‘) ppm.
Figure 2: Representative images (n = 5/mouse) obtained by confocal microscope of coronal sections of striatum (STR) and cortex (CTX) of mice (n = 3 mice/NP type) obtained 4 hours after intraperitoneal injection of 1.2 mg of the indicated NPs. (A, B) Mice treated with col-BUT NPs. (C, D) Mice treated with col NPs. (E, F) Mice treated with Col-BUT-Glu NPs. (G, H) Mice treated with Col-Phe-BUT NPs. (I, J) Mice treated with BODIPY-BUT NPs.
Figure 3: Representative images (n = 5/mouse) obtained by confocal microscope of coronal sections of striatum (STR) and cortex (CTX) of mice (n = 3 mice/NP type) obtained 4 hours after intraperitoneal injection of 1.2 mg of the indicated NPs. (A, B) Mice treated with PLGA-BUT NPs.
Figure 4: Representative images (n = 5/mouse) obtained by confocal microscope of coronal sections of striatum (STR) and cortex (CTX) of mice (n = 3 mice/NP type) obtained 4 hours after intraperitoneal injection of 1.2 mg of the indicated NPs. (A, B) Mice treated with col-LIN NPs. (C, D) Mice treated with col NPs.
Figure 5: representative images of the organized matrix formed by the col- BUT NPs according to the present invention suspended in water (A) or of col- BUT conjugates suspended in water (B, comparison).
The terms “comprising”, “comprises” are used in the present description in the usual meaning thereof and is defined as non-exhaustive of the steps, components, ingredients, or features to which it refers.
“At least one” means that at least one type of said component is included in the preparation, or in an embodiment the preparation comprises only one type of said component, in a further embodiment two types of said component are included. For example, “at least one ligand” indicates that the preparation comprises at least one type of ligand, i.e., in an embodiment the preparation comprises a ligand which is butyric acid, in a further embodiment it is to be understood that the preparation comprises two ligands, by way of example butyric acid and linoleic acid.
In an embodiment, a nanoparticle (NP) is described comprising at least one fatty acid, or derivatives thereof, which is referred to herein as a ligand, bound on the surface of a colloidal system which comprises or consists of a constituent and, optionally, one or more active ingredients of interest.
Said fatty acid, or derivative, is bound by a chemical bond to at least one functional group which is present on one or more of the constituents of said colloidal system, so as to form a constituent-ligand conjugate.
By way of example, the carboxylic group of the fatty acid or the derivative thereof is conjugated by means of an ester bond with a hydroxyl group of the constituent, or by means of an amide bond with an amino functional group of the constituent.
In an embodiment, said functional group of the constituent or of the ligand is activated, according to methods known to those skilled in the art, in order to facilitate the conjugation reaction. By way of example, the carboxyl group is optionally activated by means of an ester with an amide or a diamide to then form an amide with an amino functional group or a carboxyl group, respectively, or the carboxyl group is activated in the form of anhydride or by means of activating agents such as EDC (1-ethyl-3-(3- dimethylaminopropyljcarbodiimide) or DCC (dicyclohexylcarbodiimide).
The colloidal system is obtained according to NP preparation methods known to those skilled in the art.
The NPs according to the present invention comprise at least one conjugate and at least one constituent and, optionally, one or more further active ingredients.
In an embodiment, said at least one conjugate is present in an amount between 3 and 25% w/w, or between 5 and 20% w/w, or between 8 and 15% w/w, preferably about 10% w/w and said at least one constituent and, optionally, one or more further active ingredients, is present in amounts between 75 and 97% w/w, or between 80 and 95% w/w, or between 85 and 92% w/w, preferably about 90% w/w.
Said conjugate consists of at least one ligand chemically bound to at least one constituent.
In an embodiment, said at least one ligand is a fatty acid, preferably a linear or branched fatty acid, optionally substituted and/or esterified, saturated or unsaturated, with a number of carbon atoms between 4 and 22 or between 4 and 18. In an embodiment, it is a short-chain fatty acid, with a number of carbon atoms between 4 and 6, or medium-chain, with a number of carbon atoms between 6 and 12, or long-chain, comprising up to 22 carbon atoms, optionally substituted and/or esterified.
In an embodiment, said fatty acid is a saturated or unsaturated linear fatty acid. In an embodiment, said fatty acid is selected from the group comprising: optionally substituted and/or esterified butyric acid, valeric acid, palmitic acid, margaric acid, stearic acid, arachic acid, palmitoleic acid, oleic acid, elaidinic acid, vaccenic acid, gadoleic acid, linoleic acid, a linoleic acid, stearidonic acid, arachidonic acid, thymnodonic acid, clupanodonic acid, cervonic acid.
In an embodiment, said fatty acid is butyric acid.
In an embodiment, said fatty acid is linoleic acid.
In an embodiment, said fatty acid is substituted with phenyl, or it is esterified with glucose.
In an embodiment, said fatty acid is 2-phenylbutyric acid (Phe-BUT).
In an embodiment, said fatty acid is butyric acid esterified with glucose (Glu- BUT).
In an embodiment, said constituent is any suitable substance for preparing NPs, in particular it is selected from the group comprising cholesterol and biodegradable and biocompatible polymers and/or copolymers, preferably poly(lactic-co-glycolic) acid (PLGA). In a preferred embodiment, said constituent is cholesterol.
Said at least one ligand which is a fatty acid or derivative is bound to said constituent by chemical bond to at least one functional group of said constituent, forming said conjugate.
In an embodiment, said conjugate comprises a ligand which is butyric acid, optionally substituted and/or esterified, or linoleic acid, optionally substituted and/or esterified, and a constituent which is cholesterol, or PLGA.
In a preferred embodiment, said conjugate is selected from the group comprising butyric acid - cholesterol (col-BUT), 2-phenylbutyric acid - cholesterol (col-Phe-BUT), butyric acid - Glucose - cholesterol (col-Glu-BUT), linoleic acid - cholesterol (col-LIN), butyric acid - PLGA (PLGA-BUT).
In an embodiment, the NPs according to the present invention comprise a conjugate and a constituent, where said conjugate is present in an amount between 3 and 25% w/w, or between 5 and 20% w/w, or between 8 and 15% w/w, preferably about 10% w/w, and said constituent is present in an amount between 75 and 97% w/w, or between 80 and 95% w/w, or between 85 and 92% w/w, preferably about 90% w/w.
In an embodiment, said NPs also comprise one or more further active ingredients selected among the active ingredients which have the target thereof in the CNS. By way of example, said one or more further active ingredients are selected among antibacterials, antivirals, psychotropic drugs, antidepressants, anticancer drugs, active ingredients for treating migraines, analgesics, antagonists, or agonists of receptors present in the CNS, antibodies, antisense oligonucleotides, hormones, diagnostic agents such as paramagnetic complexes for MRI, radionuclides for nuclear medicine or for diagnosis.
In an embodiment, said active ingredient corresponds to said constituent. In a preferred embodiment, said constituent which is also the only active ingredient is cholesterol.
The NP population according to the present invention has unique features because it is extremely homogeneous both in terms of size, which is between 230 and 350 nm, preferably between 245 and 330 nm, and of PDI (square of the standard deviation divided by the average particle diameter) which is between 0.15 and 0.22, preferably between 0.17-0.21.
The measured Z potential or electrokinetic potential (ZP) is between -10 and - 21 mV.
Such chemical-physical features of the NPs according to the present invention meet the EMA/FDA guidelines.
The present invention further relates to a method for preparing NPs comprising:
- Providing at least one ligand, which is at least one fatty acid or derivative;
- Providing at least one constituent which is any suitable substance for preparing NPs, preferably selected from the group comprising cholesterol or biodegradable and biocompatible polymers or copolymers, preferably poly(lactic-co-glycolic) acid (PLGA).
- Optionally, providing one or more further active ingredients;
- Conjugating, by chemical bond, said at least one ligand with said at least one constituent;
- Obtaining a nanometric colloidal system.
In a preferred embodiment, said nanometric colloidal system is obtained through nanoprecipitation followed by evaporation of the solvent or alternatively by fusion and emulsion, resulting in the formation of NPs with a matrix shape and structure and homogeneous sizes in the order of a few hundred nanometers.
In an embodiment, said conjugate and said constituent and, optionally said at least one further active ingredient, are dissolved in organic solvents, at a temperature between room temperature and 70°C.
Said solution comprising said conjugate and said constituent and, optionally, said further active ingredient, is slowly added to an aqueous solution comprising a surfactant, preferably Pluronic F68, where said Pluronic F68 is present in said aqueous solution in an amount between 1.5 - 5% w/V, or between 2 and 3% w/V, or about 2.5% w/V, keeping under stirring.
Where said constituent is cholesterol, said solvent is preferably selected from the group comprising MeOH and EtOH and said nanoprecipitation occurs between 35 and 70°C. Where said constituent is PLGA, said solvent is preferably acetone or acetonitrile and said nanoprecipitation occurs at room temperature.
Once the nanoprecipitation has occurred, the sample thus formulated is kept under stirring until complete evaporation of the solvent.
The present invention further relates to a NP according to the invention for use in the release of active ingredients in the CNS.
In an embodiment, said use is for treating diseases related to an altered metabolism of cerebral cholesterol. In an embodiment, said use is for treating neurological, neurodegenerative diseases, selected for example from Parkinson's, Alzheimer’s, Huntington’s, aging, and cognitive decline associated therewith. In an embodiment, said use is for treating Huntington’s disease.
Advantageously, the NPs according to the present invention, in the embodiment where said constituent is cholesterol and cholesterol is also the only active ingredient charged thereon, comprise an amount of cholesterol which is near 100% by weight of the NP itself. Surprisingly, the NPs according to the present invention effectively cross the blood-brain barrier, in addition to showing good stability features, are therefore capable of delivering a high amount of cholesterol within the CNS, overcoming the problems encountered with the systems currently available based on NPs of an insufficient amount of cholesterol capable of being delivered to the CNS.
The following examples are purely for the purpose of better clarifying the invention, they are not to be understood in any way as limiting the scope thereof, the scope of protection of which is defined by the claims.
Examples
Example 1 : synthesis of the butyric acid-cholesterol conjugate
The butyric acid-cholesterol conjugate was synthesized by direct esterification of the hydroxyl group of cholesterol with butyric anhydride in the presence of triethylamine as a base (Diagram 1 ).
Figure imgf000010_0001
Diagram 1. Reagents and conditions: a) cholesterol (1.0 equiv), butyric anhydride (10 equiv), triethylamine (1.2 equiv), N2, 50°C, 18 hours, yield 94%.
Cholesterol (500 mg, 1.30 mmol; 1 eq.), butyric anhydride (2,135 ml, 13.0 mmol, 10 eq.) and triethylamine (216 pl, 1.56 mmol, 1.2 eq.) are introduced into a Schlenk tube at room temperature and under nitrogen atmosphere. The mixture was heated at 50°C for 18 h. After cooling to room temperature, the solid formed was dissolved in ethyl acetate. The organic phase was washed in succession with a saturated solution of Na2COs, 2N HCI and finally with a saturated solution of NaCI. The organic phase was dried on anhydrous Na2SC and concentrated. The reaction crude was purified by silica gel chromatography (crude/silica gel ratio 1/100) using a 9:1 (v/v) cyclohexane/ethyl acetate mixture as mobile phase. 558 mg (94% yield) of the product of interest were obtained as a waxy white solid. TLC: cyclohexane/ethyl acetate 9:1 (v/v), Rf = 0.79.
The identity and purity of the butyric acid-cholesterol conjugate was confirmed by 1H-NMR and 13C-NMR spectroscopy (Bruker 400 with 1H at 400.134 MHz and 13C at 100.62 MHz). The chemical shifts of the proton refer to the residual peaks of the solvent (CDCIs 5 = 7.26 ppm). The chemical shifts are expressed in parts per million (ppm, 5). The coupling constants are reported in hertz (Hz). The multiplicity of the signals is reported as s (singlet); d (doublet); t (triplet); q (quartet); dd (double doublet); m (multiplet); b (broad). The complete 1H and 13C NMR assignment for the butyric acid-cholesterol conjugate below confirms the chemical structure of the synthesized molecule.
Mp < 40°C. 1H-NMR (400 MHz, Chloroform-d) 50.70 (s, 3H), 0.89 (dd, J = 1 .8, 6.6 Hz, 6H), 0.91 - 1.63 (m, 30H), 1.67 (sxt, J = 7.4 Hz ), 1.86 (tdd, J = 4.4, 7.7, 15.3 Hz, 3H), 2.01 (ddt, J = 4.3, 16.7, 20.1 Hz, 2H), 2.25 (t, J = 7.4 Hz, 2H), 2.33 (dd, J = 1 .7, 7.3 Hz, 2H), 4.57 - 4.72 (m, 1 H), 5.39 (dt, J = 1 .7, 3.3 Hz, 1 H). 13C NMR (101 MHz, Chloroform-d) 5 11.86, 13.64, 18.55, 18.72, 19.32, 21.04, 22.56, 22.81 , 23.83, 24.29, 27.83, 28.01 , 28.28, 31.88, 31.91 , 35.79, 36.19, 36.61 , 37.01 , 38.17, 39.52, 39.75, 42.32, 50.04, 56.15, 56.70, 73.67, 122.56, 139.73, 173.11.
The successful conjugation between butyric acid and cholesterol is demonstrated by the 1 :1 ratio of the peak areas at 4.58-4.66 ppm (H-3) and 2.25 ppm (H-2‘) (Figure 1 ).
Example 2: Synthesis of the 2-phenylbutyric acid-cholesterol conjugate
The 2-phenylbutyric acid-cholesterol conjugate was synthesized by adapting the protocol used in example 1 as follows (diagram 2).
Figure imgf000012_0001
Diagram 2. Reagents and
Figure imgf000012_0002
a) cholesterol (1.0 equiv), 2-phenylbutyric anhydride (2 equiv), triethylamine (1.2 equiv), anhydrous DCM, N2, 50°C, 24 hours, yield 89%.
In detail, cholesterol (667 mg, 1.72 mmol, 1 eq.), 2-phenylbutyric anhydride (1 .0 mL, 3.45 mmol, 2 eq.) and triethylamine (286 pL, 2.06 mmol, 1 .2 eq.) were solubilized in anhydrous DCM inside a Schlenk tube at room temperature and under nitrogen atmosphere. The tube was sealed, and the mixture stirred at 50°C for 24 hours. After cooling to room temperature and dilution with ethyl acetate, the organic phase was washed in succession with a saturated solution of Na2COs, 2N HCI and a saturated solution of NaCI, then dried on anhydrous Na2SO4 and concentrated. The crude obtained was purified by chromatography on silica gel (crude/silica gel ratio 1 :100) using a mixture of cyclohexane/ethyl acetate 9: 1 (v/v) as mobile phase. 815 mg (89% yield) of the product of interest was obtained as a white waxy solid. TLC: cyclohexane/ethyl acetate 9: 1 , Rf = 0.85. As for the compound of example 1 , the identity and purity of the phenylbutyric acid-cholesterol conjugate were confirmed by 1H-NMR and 13C-NMR spectroscopy (Bruker 400 with 1H at 400.134 MHz and 13C at 100.62 MHz).
Pf < 60°C. 1H-NMR (400 MHz, Chloroform-d) 5 0.69 (s, 3H), 0.85 - 1.65 (m, 36H), 1.73-1.91 (m, 4H), 1.93-2.07 (m, 2H), 2.1 1 (dt, J = 7.5, 13.6 Hz, 1 H), 2.23 (d, J = 8.1 Hz, 1 H), 2.34 (d, J = 8.0 Hz, 1 H), 3.44 (dd, J = 7.2, 8.2 Hz, 1 H), 4.63 (dtd, J = 4.3, 8.6, 11 .7 Hz, 1 H), 5.37 (ddd, J = 1 .8, 5.3, 17.7 Hz, 1 H), 7.23- 7.37 (m, 5H). 13C NMR (101 MHz, Chloroform-d) 5 11 .85, 12.19, 18.72, 19.33, 21.03, 22.56, 22.82, 23.84, 24.28, 26.89, 27.54, 27.80, 28.01 , 28.23, 31.86, 31.91 , 35.79, 36.19, 36.59, 36.99, 37.83, 38.11 , 39.52, 39.73, 42.31 , 50.01 , 53.76, 56.14, 56.69, 74.12, 122.60, 127.00, 127.91 , 128.45, 139.42, 139.68, 173.43. Example 3: Synthesis of the linoleic acid - cholesterol conjugate
Cholesteryl linoleate (PL02) was synthesized by direct esterification of the cholesterol hydroxyl group with linoleic acid using EDC as activating agent for carboxylic acid and dimethylaminopyridine (DMAP) as catalyst, as shown in diagram 3:
Figure imgf000013_0001
Diagram 3. Reagents and conditions: a) linoleic acid (1.8 equiv), EDC HCI (2.5 equiv), DMAP (10% by mol), anhydrous DCM, N2, 0°C to RT, 18 hours, yield 85%.
EDC HCI (624 mg, 3.25 mmol, 2.5 equiv), DMAP (16 mg, 0.13 mmol, 0.1 equiv) and cholesterol (500 mg, 1.30 mmol, 1 equiv) were added to a solution of linoleic acid (728 l, 2.35 mmol, 1 .8 equiv) in anhydrous DCM at 0°C and under nitrogen atmosphere. The reaction was stirred at room temperature for 18 hours. The solvent was then removed, and the residue was dissolved in ethyl ether. The organic phase was washed with a saturated solution of Na2COs, 10% HCI and brine. The organic phase was dried on anhydrous Na2SO4 and concentrated. The crude was purified on silica gel using hexane/AcOEt 9:1 to give 700 mg (yield 85%) of a white waxy solid.
The identity and purity of the white waxy solid were confirmed by spectroscopy. Pf < 50°C. 1H-NMR (400 MHz, Chloroform-d) 5 0.70 (s, 3H, H-18), 0.89 (dd, J = 1.8, 6.6 Hz, 6H), 0.91 - 1.63 (m, 30H), 1.67 (sxt, J = 7.4 Hz, 2H), 1.86 (tdd, J = 4.4, 7.7, 15.3 Hz, 3H), 2.01 (ddt, J = 4.3, 16.7, 20.1 Hz, 2H), 2.25 (t, J = 7.4 Hz, 2H ), 2.33 (dd, J = 1 .7, 7.3 Hz, 2H), 4.57 - 4.72 (m, 1 H), 5.39 (dt, J = 1.7, 3.3 Hz, 1 H). 13C NMR (100 MHz, Chloroform-d) 5 11.96, 14.11 , 18.89,
19.37, 21.13, 22.65, 22.68, 23.93, 24.56, 25.13, 25.67, 27.17, 27.23, 28.17,
28.38, 29.02, 29.05, 29.13, 29.43, 31.53, 32.06, 32.10, 32.1 1 , 34.78, 35.84,
36.19, 36.77, 37.06, 38.40, 39.41 , 39.79, 42.48, 50.23, 56.50, 56.80, 73.85,
122.68, 128.37, 128.42, 130.09, 130.18, 139.92, 173.48.
Example 4: synthesis of the glucose-butyric acid-cholesterol conjugate
The synthesis of the butyric acid - fatty acid derivatized cholesterol conjugate with a glucose was performed as shown in diagram 4. To allow the simultaneous bond of cholesterol and glucose to the butyric acid, this was substituted with a succinate which maintains the same size and properties as butyric acid but has two functional groups which are exploited to bind the constituent and glucose. The cholesterol was reacted with succinic anhydride, in the presence of triethylamine as a base and a catalytic amount of DMAP in anhydrous toluene at 60°C overnight, to obtain the cholesterol hemisuccinate PLF09. In parallel, the glucose hydroxyl groups were first fully protected as trimethylsilyl ether (PLF07) by reaction with trimethylsilyl chloride and hexamethyldisilazane in pyridine at room temperature for 2 days. PLF07 was selectively deprotected to the primary hydroxy using ammonium acetate according to the procedure described by Cui et al. The coupling of PLF08 with PLF09 using DCC as a reactive activating the carboxylic functionality of the cholesterol hemisuccinate and DMAP as a catalyst in anhydrous DCM at 60°C for 2 hours, provided the conjugate PLF10 which was finally further deprotected by the trimethylsilyl groups on the glucose fraction with TFA to give the final glucose-butyric acid-cholesterol conjugate (PLF12).
Figure imgf000014_0001
Diagram 4: Reagents and conditions: a) cholesterol (1 equiv.), succinic anhydride (1.6 equiv.), triethylamine (2 equiv.), DMAP (cat.), anhydrous toluene, 60°C, overnight, yield 87%; b) glucose (1 equiv.), TMSCI (6 equiv.), hexamethyldisilazane (6 equiv.), pyridine, 0°C at rt, 2 days, yield 93%; c) ammonium acetate (2 equiv.), DCM: MeOH 1 :1 v/v, rt, 6 h, quantitative yield; d) PLF09 (1 equiv.), PLF08 (1 equiv.), DCC (1.3 equiv.), DMAP (cat.), anhydrous THF, N2, 60°C, 2 hours, yield 62%; e) TFA (22 equiv.), DCM, 0°C, 1 h, 51% yield. Synthesis of cholesterol hemisuccinate (PLF09)
A solution of cholesterol (1.0 g, 2.40 mmol, 1 equiv.), succinic anhydride (385 mg, 3.84 mmol, 1.6 equiv), triethylamine (80 uL, 0.6 mmol, 0.25 equiv) and DMAP (cat.) in anhydrous toluene and under an inert atmosphere is heated at 60°C overnight. The reaction mixture is then diluted with DCM and the organic phase is washed with 1 N HCI: the organic phase is dried with anhydrous Na2SO4 and concentrated. The product is obtained by crystallization from cyclohexane (1.09 g, yield 87%) as a waxy white solid. Pf 173-176 °C. 1H NMR (400 MHz, Chloroform-d) 5 0.50-2.35 (m, 43H), 2.62 (t, 2H), 2.69 (t, 2H), 4.63 (m, 1 H), 5.38 (d, 1 H).
Synthesis of penta-O-Trimethylsil-a-D-glucopyranose (PLF07)
A mixture of trimethylchlorosilane (4.23 mL, 33.4 mmol, 6 equiv.) and hexamethyldisilazane (2.34 mL, 11.1 mmol, 2 equiv.) was slowly added to a solution of D-glucose (1 , 0 g, 5.56 mmol, 1 equiv.) in pyridine (5 mL) at 0°C and under argon atmosphere, and with violent stirring. The temperature was slowly brought to room temperature and the reaction left under stirring for 2 days. The excess solvent and reagents were evaporated under reduced pressure. The residue was dissolved in ethyl ether, and the organic phase was washed in succession with saturated solution of NaHCCh, and a solution of CuSO4 at 15%. The organic phase was dried on anhydrous Na2SO4 and concentrated to give 2.8 g of colorless syrup (yield 93%).
1H NMR (400 MHz, Chloroform-d) 5 -0.13 - 0.09 (m, 45H), 3.12 - 3.30 (m, 2H), 3.47 - 3.67 (m, 4H), 4.86 (d, J = 3.1 Hz, 1 H).
Synthesis of 1,2,3,4-tetra-O-Trimethylsilyl-a-D-glucopyranose (PLF08) Ammonium acetate (800 mg, 10.35 mmol, 2 equiv.) was added to a solution of PLF07 (2.8 g, 5.18 mmol, 1 equiv.) in DCM: MeOH 1 :1 v/v (30 mL), and the mixture was mixed for 12 hours at room temperature. The reaction solvent was removed, and the residue was suspended in diethyl ether. The organic phase was washed with water, dried on anhydrous Na2SO4 and concentrated to obtain 2.6 g (quantitative yield) of a colorless syrup, pure enough to be used in the next step without further purification.
1H NMR (400 MHz, Chloroform-d) 50.14-0.19 (m, 36H), 3.34 (dd, 1 H, J1 = 2.8 Hz, J2 = 9.2 Hz), 3.66 (t, 1 H, J = 9.2 Hz), 3.68 (dd, 1 H, J1 = 4.8 Hz, J2 = 12 Hz), 3.73-3.76 (m, 2H), 3.80 (t, 1 H, J = 8.8 Hz), 5.16 (d, 1 H, J = 3.2 Hz).
Synthesis of 1,2,3,4-tetra-O-Trimethylsilyl-a-D-glucopyranosyl cholesterol succinate (PLF10)
DCC (60 mg, 0.26 mmol, 1.3 eq.) and DMAP (cat.) were added to a solution of PLF08 (100 mg, 0.20 mmol, 1 equiv.) in anhydrous DCM (5 mL), PLF09 (101 mg, 0.20 mmol, 1 equiv.), at room temperature and under nitrogen atmosphere. The mixture was left to react under the same conditions for 2 days. The solvent was evaporated and the crude rapidly purified by flash chromatography (mobile phase CE:AcOEt 7:3) to obtain 120 mg (62% yield) of a colorless liquid.
1H NMR (400 MHz, chloroform-d) 5 -0.00 (dd, J = 3.8, 5.8 Hz, 36H), 0.52 (s, 3H), 0.71 (dd, J = 1.7, 6.6 Hz, 6H), 0.76 (d, J = 6.5Hz, 3H), 0.86 (s, 3H), 0.97 (td, J = 7.0, 14, 0, 14.9 Hz, 4H), 1.34 (d, J = 52.0 Hz, 16H), 1.63 - 1.90 (m, 6H),
2.16 (d, J = 7.9 Hz, 2H), 2.46 (d, J = 6.6 Hz, 2H), 2.52 (dd, J = 4.0, 9.0 Hz, 2H),
3.16 - 3.40 (m, 2H), 3.58 - 3.83 (m, 2H), 3.91 (dd, J = 5.4, 11 .8 Hz, 1 H), 4.21 (dd, J = 2.3, 11 .8 Hz, 1 H), 4.47 (d, J = 10.3 Hz, 1 H), 4.85 (d, J = 3.0 Hz, 1 H) , 5.21 (d, J = 5.1 Hz, 1 H). ESI-MS m/z [M+H]+ calculated for C49H93O9Si4+: 937.6; found: 937.7.
Synthesis of a D-glucopyranosyl cholesterol succinate (PLF12) Trifluoroacetic acid (0.20 mL, 2.81 mmol, 22 equiv.) was added to a solution of PLF10 (120 mg, 0.13 mmol, 1 equiv.) in DCM (5 mL) at 0°C. The reaction was stirred under the same conditions for 1 hour and then concentrated. The residue was triturated on diethyl ether and the precipitate, decanted by centrifugation, was washed with ethyl ether and dried to give 43 mg of the glucose-butyric acid-cholesterol conjugate with the appearance of a white solid (yield of 51 %).
The identity and purity of the conjugate were confirmed by spectroscopy. Pf 120°C with decomposition. 1H NMR (400 MHz, DMSO-d6) 50.66 (s, 3H), 0.85 (dd, J = 1.8, 6.6 Hz, 6H), 0.90 (d, J = 6.5 Hz, 3H), 0.95 - 1.21 (m, 8H), 1.19 - 1 .61 (m, 8H), 1 .69 - 2.03 (m, 6H), 2.26 (d, J = 8.1 Hz, 2H), 2.45 - 2.61 (m, 8H), 2.91 (t, J = 8.3 Hz , 1 H), 3.04 (dt, J = 6.5, 13.2 Hz, 1 H), 3.13 (ddd, J = 2.5, 6.1 , 8.9 Hz, 1 H), 3.32 (dd, J = 6.4, 9.7 Hz, 1 H), 3.43 ( t, J = 9.1 Hz, 1 H), 3.78 (dd, J = 6.5, 10.0 Hz, 1 H), 3.98 (td, J = 6.5, 11.9 Hz, 1 H), 4.22 - 4.36 (m, 2H), 4.47 (d , J = 9.2 Hz, 1 H), 4.90 (d, J = 3.7 Hz, 1 H), 5.35 (d, J = 4.8 Hz, 1 H). 13C NMR (101 MHz, DMS0-d6) 5 171.93, 171.72, 139.28, 121.70, 95.76, 75.02, 74.26, 73.07, 72.85, 69.30, 63.23, 55.46, 55.31 , 49.42, 41.56, 39.00, 38.44, 37.59, 36.07, 35.87, 35.42, 34.89, 31.23, 31.06, 28.88, 28.55, 28.13, 28.11 , 27.33, 23.79, 23.23, 21.79, 20.29, 18.55, 18.06, 11.11.
Example 5: synthesis of the butyric acid-PLGA conjugate
In order to avoid the polyacylation which would occur due to the direct reaction of butyric acid with the free hydroxyls of the PLGA, a diethylenediamine was inserted between the ligand and the constituent which allowed selectively binding the free carboxy groups of the butyric acid and PLGA by means of amide bond. The synthesis of the butyric acid-PLGA conjugate is shown in diagram 5. Ethylenediamine hydrochloride, mono protected to a nitrogen atom by a removable group such as Cbz, was reacted with butyryl chloride, in the presence of triethylamine as a base, in anhydrous DCM at room temperature overnight, to obtain the amide intermediate PLF13. The protective group on the second nitrogen atom was then removed by hydrogenolysis using sodium borohydride as a hydrogen source and catalyzed by 10% Pd/C. The reaction was carried out in methanol at room temperature for 4 hours to provide the amine intermediate PLF14 with quantitative yield.
Finally, the coupling of PLF14 with the PLGA was carried out using EDC and HOBt as reactants activating the carboxyl functionality of the PLGA and trimethylamine as a base in anhydrous DCM at room temperature for 18 hours. By means of purification by crystallization, the butyric acid - PLGA conjugate was obtained with a yield of 65%.
Figure imgf000017_0001
PLF15 Diagram 5. Reagents and conditions, a) N-Cbz-ethylenediamine (1 equiv), butyryl chloride (2 equiv), triethylamine (3 equiv), anhydrous DCM, N2, room temperature, 18 hours, quantitative yield; b) PLF13 (1 equiv), 10% Pd/C (0.1 equiv), NaBH4 (1 equiv), methanol, room temperature, 4 hours, quantitative yield; c) PLF14 (320 mg, 1 equiv), PLGA (320 mg), EDC HCI (1 equiv), HOBt (1 equiv), triethylamine (2 equiv), anhydrous DCM, room temperature, 18 hours, yield 65%.
Synthesis of benzyl-(2-butyramidoethyl)carbamate (PLF13)
Triethylamine (905 uL, 6.51 mmol, 3 equiv) and butyryl chloride (451 uL, 4.35 mmol, 2 equiv) are added to a suspension of N-Cbz-ethylenediamine hydrochloride (500 mg, 2.17 mmol, 1 equiv) in anhydrous DCM at room temperature and under nitrogen atmosphere. It is left to react under the same conditions for 18 hours, after which the solvent is evaporated. The residue is taken up with ethyl acetate and the organic phase is washed in succession with 10% HCI, a saturated solution of Na2COs and finally with a saturated solution of NaCI. The organic phase is dried on anhydrous Na2SO4 and concentrated. 560 mg (98% yield) of a white solid are obtained. The spectral characterization is consistent with the literature data.
Synthesis of N-(2-aminoethyl)butyramide (PLF14)
10% Pd/C (10% by weight, 46 mg) and sodium borohydride (66 mg, 1.74 mmol, 1 equiv) are added to a solution of PLF13 (460 mg, 1.74 mmol, 1 equiv) in methanol and at room temperature. It is left to react under the same conditions for 4 hours, after which the reaction mixture is filtered on celite and concentrated. It is purified by means of a cationic resin (Amberlyst 15) to obtain 200 mg (quantitative yield) of a white solid. 1H NMR (400 MHz, Methanol-d4) 5 0.96 (t, J = 7.4 Hz, 3H), 1 .57 - 1 .75 (m, 2H), 2.18 (t, J = 7.5 Hz, 2H), 3.33 (p, J = 1.6 Hz, 4H).
Synthesis of butyric acid - PLGA conjugate (PLF15)
EDC HCI (536 mg, 2.75 mmol, 1 equiv), HOBt (371 mg, 2.75 mmol, 1 equiv), PLF14 (320 mg, 2.75 mmol, 1 equiv) and triethylamine (763 uL, 5.50 mmol, 2 equiv.) are added to a solution of PLGA (320 mg) in anhydrous DCM at room temperature and under an inert atmosphere. It is left to react under the same conditions for 18 hours, after which the solvent is removed by filtration. The crude is triturated on diethyl ether and the precipitate discarded. The filtrate is concentrated and ground on methanol. A white solid precipitates which is recovered by centrifugation, washed with methanol and DCM and concentrated. 210 mg of a white solid are obtained. The identity and purity of the conjugate were confirmed by NMR and IR spectroscopy. 1H NMR (400 MHz, chloroform-d) 5 1.08 (t, J = 7.2 Hz, 3H, CH3-But), 1.46 - 1.68 (m, 78H, CHs-PLGA), 1 .94 (p, J = 6.8 Hz, 2H), 2.09 - 2.23 (m, 2H), 3.46 (q, J = 7.1 Hz, 5H), 4.60 - 4.96 (m, 52H, CH2-PLGA), 5.21 (tq, J = 21 .2, 8.9, 7.6 Hz, 26H, CH- PLGA).
Example 6: NP preparation col-BUT NPs comprising butyric acid as ligand and cholesterol as constituent and sole active ingredient were prepared as follows by nanoprecipitation. The cholesterol-butyric acid conjugate prepared as in example 1 and cholesterol were provided. In order to trace the NPs after administration, a labeled cholesterol was also provided, i.e. , cholesterol-Cy5 (Invitrogen) or BODIPY cholesterol. The components were dissolved in MeOH at 60°C. The following ratios were used: 10% w/w butyric acid-cholesterol conjugate, 85% w/w cholesterol, 5% w/w cholesterol-Cy5 (or cholesterol-Bodipy). The hot solution was slowly added to an aqueous solution of Pluronic F68 (2.5% w/V) kept under stirring. Once the nanoparticles were formed, the stirring was continued until the organic solvent evaporated.
Following the same nanoprecipitation protocol, col-Phe-BUT NPs were obtained, where the conjugate is phenylbutyric acid (Phe-BUT) conjugated with cholesterol, col-Glu-BUT NPs, where the conjugate is glucose-modified butyric acid conjugated with cholesterol and col-LIN NPs where the conjugate is linoleic acid conjugated with cholesterol.
Said NPs are obtained with the same constituent which is cholesterol at the same concentrations and under the same experimental conditions described above for the col-BUT NPs.
PLGA-BUT NPs were obtained by nanoprecipitation starting from butyric acid as ligand and PLGA (PM about 11 ,000, lactic/glycolic ratio 50:50) as constituent. In this embodiment, the components were provided in the following % w/w ratios: 10% PLGA-BUT, 88% PLGA, 2% PLGA-Cy5. The NPs were obtained after solubilization of the components in acetone and subsequent addition to an aqueous solution at room temperature of Pluronic F68 (2.5% w/v) and kept under stirring. Once the NPs formed, the stirring was continued until the organic solvent evaporated.
Example 7: NP morphological characterization
A chemical-physical analysis and photon correlation spectroscopy (PCS) analysis were carried out to define the average size of the col-BUT NPs obtained in example 6 in aqueous suspension and the surface charge thereof. A monomodal population was thus highlighted, the NPs are homogeneous with a low polydispersity index and the population is characterized by sizes between 250 and 350 nm and a surface charge potential of about -15 mV.
To better define the NPs’ structure and morphology, which characterize them by defining the peculiar technological-pharmaceutical properties thereof, a microscopic analysis was carried out with SEM-FEG transmission microscope. col-BUT NPs obtained as in example 6 were suspended in MilliQ water and observed under transmission electron microscope. The exemplary photographs in Figure 5A show that they form a matrix structure, organized in nanometer-sized particles, thus defining a new nanometric matrix system.
For comparative purposes, the conjugates of example 1 and 2, not formulated as in example 6 but directly suspended in MilliQ water, were analyzed. As is clear from Figure 5B, said conjugates are not organized in a defined structure, they are configured as disorganized lipid chains.
A quantitative characterization of the col-BUT NPs obtained in example 6 was then carried out. The mean size, the polydispersity index (PDI) and the zeta potential or electrokinetic potential (ZP) were measured. The measurements were made as follows:
- POI: PCS analysis (Photon Correlation Spectroscopy), using a Zetasizer Nano ZS (Malvern, UK; Laser 4 mW He - Ne, 633 nm, automatic laser attenuator, 100-0.0003% transmission, Avalanche Detector photodiode, QE> 50% at 633 nm, T = 25 ° C) . The samples were diluted to a final concentration of ~ 0.1 mg/mL before being tested. The data are expressed as the average of at least ten independent preparations.
- Zeta potential: Zetasizer Nano ZS (Malvern) with a combination of laser Doppler velocimetry and the M3-PALS phase analysis light scattering method. The same samples subjected to PCS (0.1 mg/mL) were analyzed using DTS1070 zeta-potential cuvettes and expressed as the average of at least ten independent preparations.
The data obtained on 10 independent samples, carrying out 5 analyses in triplicate per sample, gave the following results:
- Size: 287 nm, Std Dev 38
- PDI: 0.19, Std Dev 0.02
- Zeta potential: -15.7 Std Dev 4.2 Example 8: passage across the BBB
To evaluate the passage of the BBB in vivo, an aqueous suspension of col- BUT NPs obtained as in example 6, corresponding to 1.2 mg of NPs, was injected into mice intraperitoneally. After 4 hours, the mice were sacrificed to obtain brain sections (15 pm thick) to be subjected to microscopic analysis as described in the literature (Vilella A. et al. J. Control. Rel. 2014; 174:195-201 ). The marked cholesterol allowed to highlight the presence thereof in the photomicrographs under confocal microscope, some representative images are shown in Figure 2. The data shown are examples of photographs obtained from 3 animals treated for each type of nanoparticles, after having acquired 5 images for each animal. The Cy5 dye signal is highlighted by the arrows in the figure. In particular, Figure 2A shows the images obtained from striatum sections, Figure 2B from cortex sections of mice treated with col-BUT NPs. For comparative purposes, Figures 2C and 2D show a representative image of what was obtained with NPs consisting of cholesterol and dye, therefore without the ligand, in striatum and cortex, respectively. No passage of the blood-brain barrier is observed. Vice versa, Figures 2A and 2B, according to the invention, show the presence of NP signals inside the cerebral parenchyma, unequivocally signifying the passage across the BBB observed with col-BUT NPs. Similar results were shown both in the striatum and in the cortex for each type of tested NP according to the present invention. By way of example, col-BUT-Glu NPs (Figure 2E striatum and 2F cortex), col-Phe-BUT NPs (Figure 2G striatum and 2H cortex) were tested. The ability of the NPs according to the present invention to cross the BBB was also confirmed by using BODIPY-cholesterol (BODIPY-BUT NPs) as a marker. In detail, the signal detected in the striatum (Figure 21) and in the cortex (Figure 2J) confirms the entry of BODIPY-cholesterol into the CNS, obtained by virtue of the NPs according to the present invention. Similarly, the PLGA-BUT NPs also demonstrated the ability to cross the BBB, by virtue of the conjugation of PLGA with the ligand BUT (Figure 3A striatum and 3B cortex). In the absence of the ligand, the PLGA NPs do not reach the CNS, as widely described in the literature (Del Grosso A et al. Sci Adv. 2019; 5(11 ):eaax7462).
Finally, Figure 4 shows sections of striatum (Figure 4A) and cortex (Figure 4B) of mice treated with NPs obtained using cholesterol conjugated with the ligand linoleic acid (col-LIN NPs) and related controls without ligand (Figure 4C and 4D).
The data described here show that the NPs according to the present invention effectively cross the BBB and are found in the cortex sections and in the striatum sections.

Claims

1. Nanoparticles (NPs) for the delivery of active ingredients across the blood-brain barrier comprising at least one ligand bound on the surface of a colloidal system, wherein said at least one ligand is at least one linear or branched fatty acid, optionally substituted and/or esterified, saturated or unsaturated, with a number of carbon atoms between 4 and 22 and said colloidal system comprises or consists of at least one constituent and, optionally, one or more active ingredients, characterized in that said ligand is bound by a chemical bond to at least one functional group which is present on one or more of the constituents of said colloidal system, so as to form a constituent-ligand conjugate.
2. Nanoparticles according to claim 1 , wherein said fatty acid has a number of carbon atoms between 4 and 18, or between 4 and 12, or between 4 and 6, or between 6 and 12.
3. Nanoparticles according to claim 1 or 2, wherein said fatty acid is a linear fatty acid, optionally substituted and/or esterified.
4. Nanoparticles according to one of claims 1 to 3, wherein said fatty acid is selected from the group comprising: optionally substituted and/or esterified butyric acid, valeric acid, palmitic acid, margaric acid, stearic acid, arachic acid, palmitoleic acid, oleic acid, elaidinic acid, vaccenic acid, gadoleic acid, linoleic acid, a linoleic acid, stearidonic acid, arachidonic acid, thymnodonic acid, clupanodonic acid, cervonic acid.
5. Nanoparticles according to one of claims 1 to 4, wherein said fatty acid is substituted with a phenyl.
6. Nanoparticles according to one of claims 1 to 5, wherein said fatty acid is esterified with glucose.
7. Nanoparticles according to one of claims 1 to 6, wherein said at least one ligand is selected from the group comprising butyric acid (BUT), linoleic acid (LIN), 2-phenylbutyric acid (Phe-BUT), butyric acid esterified with glucose (Glu-BUT).
8. Nanoparticles according to one of claims 1 to 7, wherein said at least one constituent is selected from the group comprising cholesterol, biodegradable and biocompatible polymers and/or copolymers, for example poly(lactic-co-glycolic) acid (PLGA).
9. Nanoparticles according to one of claims 1 to 8, wherein said conjugate is selected from the group comprising butyric acid - cholesterol (col- BUT), 2-phenylbutyric acid - cholesterol (col-Phe-BUT), butyric acid - glucose - cholesterol (col-Glu-BUT), linoleic acid - cholesterol (col-LIN), butyric acid - PLGA (PLGA-BUT).
10. Nanoparticles according to one of claims 1 to 9, wherein said conjugate is in an amount between 3 and 25% w/w, or between 5 and 20% w/w, or between 8 and 15% w/w, preferably about 10% w/w, said at least one constituent and, optionally, one or more further active ingredients, is in a total amount between 75 and 97% w/w, or between 80 and 95% w/w, or between 85 and 92% w/w, preferably about 90% w/w.
11. Nanoparticles according to one of claims 1 to 10, which have a diameter between 230 and 350 nm, preferably between 245 and 330 nm and a PDI between 0.15 and 0.22, preferably between 0.17 and 0.21.
12. Nanoparticles according to one of claims 1 to 11 , which have a Zeta potential between -10 and -21 mV.
13. A method for preparing NPs according to one of claims 1 to 12, comprising:
- Providing at least one ligand, which is at least one linear or branched fatty acid, optionally substituted and/or esterified, saturated or unsaturated, with a number of carbon atoms between 4 and 22;
- Providing at least one constituent selected from the group comprising cholesterol and/or biodegradable and biocompatible polymers and/or copolymers, for example poly(lactic-co-glycolic) acid (PLGA);
- Optionally, providing one or more further active ingredients;
- Conjugating, by chemical bond, said at least one ligand with said at least one constituent;
- Obtaining a nanometric colloidal system.
14. A method according to claim 13, wherein said nanometric colloidal system is obtained through nanoprecipitation followed by evaporation of the solvent, wherein said nanoprecipitation occurs by dissolving said conjugate and said constituent and, optionally, said further active ingredient in organic solvents at a temperature between room temperature and 70°C, and adding said solution comprising said conjugate and said constituent and, optionally, said further active ingredient, to an aqueous solution comprising a surfactant, keeping under stirring until the nanoprecipitation has occurred.
15. A method according to claim 14, wherein said surfactant is Pluronic F68, wherein said Pluronic F68 is present in said aqueous solution in an amount between 1.5 - 5% w/V, or between 2 and 3% w/V, or about 2.5% w/V.
16. A method according to one of claims 13 to 15, wherein said constituent is cholesterol and said solvent is selected from the group consisting of MeOH and EtOH and said nanoprecipitation occurs at a temperature between 35 and 70°C.
17. A method according to one of claims 13 to 15, wherein said constituent is PLGA and said solvent is selected from the group consisting of acetone or acetonitrile and said nanoprecipitation occurs at room temperature.
18. Nanoparticles obtained according to the method of one of claims 13 to
17.
19. Nanoparticles according to one of claims 1 to 12, or according to claim
17, for use in the release of active ingredients in the CNS.
20. Nanoparticles according to one of claims 1 to 12, or according to claim
18, wherein said constituent and active ingredient is cholesterol, for use in treating diseases related to an altered metabolism of cerebral cholesterol.
21. Nanoparticles according to one of claims 1 to 12, or according to claim 18, for use in treating neurological or neurodegenerative diseases selected in the group comprising Parkinson’s, Alzheimer's, Huntington’s, aging and cognitive decline associated therewith.
22. Nanoparticles for use according to claim 21 , wherein said disease is Huntington’s.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060002852A1 (en) * 2004-07-01 2006-01-05 Yale University Targeted and high density drug loaded polymeric materials
WO2012177986A2 (en) * 2011-06-22 2012-12-27 Vyome Biosciences Conjugate-based antifungal and antibacterial prodrugs
WO2013033513A1 (en) * 2011-08-31 2013-03-07 University Of Georgia Research Foundation, Inc. Apoptosis-targeting nanoparticles
WO2019024784A1 (en) * 2017-07-31 2019-02-07 The Chinese University Of Hong Kong Fatty acid conjugated nanoparticles and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060002852A1 (en) * 2004-07-01 2006-01-05 Yale University Targeted and high density drug loaded polymeric materials
WO2012177986A2 (en) * 2011-06-22 2012-12-27 Vyome Biosciences Conjugate-based antifungal and antibacterial prodrugs
WO2013033513A1 (en) * 2011-08-31 2013-03-07 University Of Georgia Research Foundation, Inc. Apoptosis-targeting nanoparticles
WO2019024784A1 (en) * 2017-07-31 2019-02-07 The Chinese University Of Hong Kong Fatty acid conjugated nanoparticles and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRIOSCHI ANDREA ET AL: "Cholesterylbutyrate Solid Lipid Nanoparticles as a Butyric Acid Prodrug", vol. 13, no. 2, 1 January 2008 (2008-01-01), pages 230 - 254, XP055813265, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245427/pdf/molecules-13-00230.pdf> DOI: 10.3390/molecules13020230 *
GYULAI G ET AL: "Influence of surface properties of polymeric nanoparticles on their membrane affinity", EUROPEAN POLYMER JOURNAL, PERGAMON PRESS LTD OXFORD, GB, vol. 49, no. 9, 6 March 2013 (2013-03-06), pages 2495 - 2503, XP028689520, ISSN: 0014-3057, DOI: 10.1016/J.EURPOLYMJ.2013.02.024 *
SERPE L: "Cholesteryl butyrate solid lipid nanoparticles as a butyric acid pro-drug: effects on cell proliferation, cell-cycle distribution and c-myc expression in human leukemic cells", ANTICANCER DRUGS, 30 June 2004 (2004-06-30), XP055813268, Retrieved from the Internet <URL:https://journals.lww.com/anti-cancerdrugs/Abstract/2004/06000/Cholesteryl_butyrate_solid_lipid_nanoparticles_as.15.aspx> [retrieved on 20210614] *

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