WO2024095144A1 - Lipidic nanoparticles comprising an encapsulated drug and method of producing the same - Google Patents

Lipidic nanoparticles comprising an encapsulated drug and method of producing the same Download PDF

Info

Publication number
WO2024095144A1
WO2024095144A1 PCT/IB2023/060950 IB2023060950W WO2024095144A1 WO 2024095144 A1 WO2024095144 A1 WO 2024095144A1 IB 2023060950 W IB2023060950 W IB 2023060950W WO 2024095144 A1 WO2024095144 A1 WO 2024095144A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipids
levodopa
lipidic nanoparticles
previous
nanoparticles
Prior art date
Application number
PCT/IB2023/060950
Other languages
French (fr)
Inventor
Maria DO CARMO DA SILVA PEREIRA
Joana Angélica DE SOUSA LOUREIRO
Débora Sofia SPÍNOLA NUNES
Maria João ALVES RAMALHO
Stéphanie MACHADO ANDRADE
Original Assignee
Universidade Do Porto
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidade Do Porto filed Critical Universidade Do Porto
Publication of WO2024095144A1 publication Critical patent/WO2024095144A1/en

Links

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
    • A61K9/48—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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • 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
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs

Definitions

  • This application relates to lipidic nanoparticles comprising an encapsulated drug and a method of producing the same. It also relates to a pharmaceutical composition comprising the lipidic nanoparticles.
  • Parkinson's Disease is the second most prevalent neurodegenerative disease, characterized by a reduction in dopamine levels in the brain, caused by a loss of nerve cells in substantia nigra, leading to movement problems once dopamine is responsible for regulating the movement of the body.
  • the continuous increase of PD rate demands for a fast development of preventive and/or curative therapeutic strategies that are truly effective.
  • Levodopa is used as dopamine replacement agent for PD treatment since it is converted in dopamine when absorbed by the nerve cells in the brain, which can restore functional movements.
  • This medication doesn't slow down or cure PD, but can control symptoms like tremors, rigidity of the muscles and bradykinesia.
  • long-term treatment with levodopa is associated with physical side effects like dyskinesia and fluctuations in motor response, called on/off phenomena of PD, related with the intermittent delivery of levodopa to the brain due to drug level fluctuations in plasma.
  • BBB blood brain barrier
  • the conventional PD therapies are available as oral pills and transdermal patches.
  • long-term treatment with levodopa is associated with physical side effects like dyskinesia and fluctuations in motor response, called the wearing off of medication effect, related with the intermittent delivery of levodopa to the brain due to fluctuations of drug in plasma levels.
  • the wearing off of medication effect related with the intermittent delivery of levodopa to the brain due to fluctuations of drug in plasma levels.
  • After 5 years of levodopa intake approximately 40% of PD patients experience motor fluctuations and/or dyskinesias. The number increases to 90% after 9 or more years of therapy with levodopa [1].
  • dyskinesia this side effect is developed in about 63% and 34% of PD patients after 2 and 5 years of therapy, respectively [2,3].
  • the present invention relates to lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases, wherein the nanoparticles are solid lipid nanoparticles or are nanostructured lipid carriers composed of a mixture of solid lipids and liquid lipids.
  • the encapsulated drug is selected from levodopa, a levodopa/carbidopa combination, dopamine agonists, monoamine oxidase-B inhibitors, or amantadine.
  • the solid lipids are selected from glyceryl distearate, behenoyl polyoxyl-8 glycerides, stearoyl polyoxyl-32 glycerides, mono- or di- or triglyceride esters of fatty acids C8 to C18, glyceryl dibehenate, stearic acid, hydrogenated coco-glycerides, PEG-8 beeswax, cetyl palmitate, hydrogenated palm oil, glyceryl tristearate, glyceryl monostearate, or their mixtures.
  • the liquid lipids are selected from mediumchain triglyceride ester of saturated coconut/palmkernel oil derived caprylic and capric fatty acids and plant derived glycerol, vitamin E, decyl oleate, isopropyl myristate, medium-chain triglycerides of caprylic (C8) and capric (CIO) acids, cetyl dimeticone, or their mixtures.
  • the solid lipids are present in a weight percentage from 0.1 to 99.9 % in the mixture of solid lipids and liquid lipids, the remaining quantity being liquid lipids.
  • the nanoparticles comprise an average size between 100 nm and 200 nm.
  • the drug is encapsulated in the lipidic nanoparticles in a concentration between 0.01 and 30 mg/mL.
  • the surface of the lipidic nanoparticles is modified with proteins or peptide, or antibodies.
  • the neurologic or chronic diseases are selected from epilepsy, Alzheimer's and other dementias, strokes, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, neurological infections, brain tumors, major depression, schizophrenia, attention deficit hyperactivity disorder or Autism.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases.
  • the present invention also relates to a method of producing the lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases, comprising the following steps:
  • the surfactant is selected from poloxamer 407, poloxamer 188, poloxamer 182, poloxamer 908, egg lecithin, soy lecithin, polysorbate 20, polysorbate 60, or polysorbate 80.
  • the surfactant is used in a concentration between 1 and 10%.
  • the present invention relates to biocompatible and biodegradable lipidic nanoparticles (NPs) comprising an encapsulated drug for the treatment of a neurologic or a chronic disease, for example levodopa, that will provide a sustained and controlled release of said drug to increase the therapeutic effect of the drug and reduce side effects.
  • NPs biocompatible and biodegradable lipidic nanoparticles
  • the drug in neurologic diseases in the brain, the drug will be released from the NPs and will achieve effective drug levels at the required location, decreasing the side effects for the other tissues/organs. Since the selected NPs have optimal clearance characteristics, the NPs will be degraded after releasing their load, reducing potential side effects due to NPs toxicity.
  • nanoparticles have potential to be used to encapsulate different active molecules and can be applied for the treatment of other neurological or chronic diseases.
  • the presently disclosed lipidic nanoparticles present sizes lower than 200 nm, stability over several months (6 months) and encapsulation efficiencies up to 79%.
  • the present invention Compared to the previously reported nanoparticles encapsulating levodopa (poly (lactic-co-glycolic acid) (PLGA) nanoparticles and liposomes), the present invention presents several advantages. These PLGA nanoparticles and liposomes present several inadequate properties to be used in the treatment of PD, such as large size (above 200 nm) and/or low stability and/or lower encapsulation efficiencies [7 - 11]. Therefore, the present invention represents a novel approach with improved features to treat neurologic or chronic diseases .
  • PLGA poly (lactic-co-glycolic acid)
  • Neurologic diseases and chronic diseases can be selected from, but not limited to, epilepsy, Alzheimer's and other dementias, strokes, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, neurological infections, brain tumors, major depression, schizophrenia, attention deficit hyperactivity disorder, Autism.
  • a PD therapy using a drug such as levodopa encapsulated into the lipidic NPs will be increased, since the sustained and continuous release of a drug from NPs will maintain the active doses of drug, reducing the wearing off of medication effect, making therapy truly effective. Since the NPs have ability to reach the brain, there will be a significant increase of the drug at the targeted organ (brain), providing reduced systemic toxicity with less side effects, and lower doses will be required, compared to the conventional therapy.
  • a drug such as levodopa encapsulated into the lipidic NPs
  • a drug is understood as an active molecule intended for use in preventing or curing a disease or relieving pain or disease symptoms.
  • levodopa-loaded NPs were successfully produced with higher EE, using solid lipid nanoparticles (NPLs) and nanostructured lipid carriers (NLCs) as carriers.
  • NPLs solid lipid nanoparticles
  • NLCs nanostructured lipid carriers
  • the successfully loaded-levodopa formulations showed a size smaller than 200 nm, an ideal size for systemic administration and passing the BBB with a narrow-size distribution.
  • ZP values levodopa encapsulation did not significantly impact the surface charge of the NPs.
  • the ZP values of the formulation were high enough to ensure their stability after systemic administration, avoiding NPs aggregation.
  • the levodopa- loaded SLNs and levodopa-loaded NLCs remained stable and did not form aggregates over 6 months.
  • Figure 1 shows the structure of levodopa.
  • Figure 2 shows a UV-Vis Spectrum obtained for levodopa with indication of the characteristic absorbance peaks of this drug at 0.1 mg/ml (280 nm).
  • the present invention relates to lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases.
  • the nanoparticles of the present invention are of the solid lipid nanoparticles (SLN) type - composed of solid lipids - or nanostructured lipid carriers (NLC) type - composed of a mixture of solid lipids and liquid lipids.
  • SSN solid lipid nanoparticles
  • NLC nanostructured lipid carriers
  • the encapsulated drug is selected from, but not limited to, levodopa, a levodopa/carbidopa combination, dopamine agonists, monoamine oxidase-B inhibitors, or amantadine.
  • the nanoparticles comprise solid lipids selected from, but not limited to, glyceryl distearate, behenoyl polyoxyl-8 glycerides, stearoyl polyoxyl-32 glycerides, mono- or di- or triglyceride esters of fatty acids C8 to C18, glyceryl dibehenate, stearic acid, hydrogenated coco-glycerides, PEG-8 beeswax, cetyl palmitate, hydrogenated palm oil, glyceryl tristearate, glyceryl monostearate, or their mixtures.
  • solid lipids selected from, but not limited to, glyceryl distearate, behenoyl polyoxyl-8 glycerides, stearoyl polyoxyl-32 glycerides, mono- or di- or triglyceride esters of fatty acids C8 to C18, glyceryl dibehenate, stearic acid, hydrogenated coco-gly
  • the solid lipids are present in a weight percentage from 0.1 to 99.9 %. The remaining quantity being liquid lipids.
  • the nanoparticles comprise solid lipids selected from, but not limited to, glyceryl distearate, behenoyl polyoxyl-8 glycerides, stearoyl polyoxyl-32 glycerides, mono- or di- or triglyceride esters of fatty acids (C8 to C18), glyceryl dibehenate, stearic acid, hydrogenated coco-glycerides, PEG-8 beeswax, cetyl palmitate, hydrogenated palm oil, glyceryl tristearate, glyceryl monostearate, or their mixtures.
  • liquid lipids are selected from, but not limited to, medium-chain triglyceride ester of saturated coconut/palmkernel oil derived caprylic and capric fatty acids and plant derived glycerol, vitamin E, decyl oleate, isopropyl myristate, medium-chain triglycerides of caprylic (C8) and capric (CIO) acids, cetyl dimeticone, or their mixtures.
  • the nanoparticles comprise an average size between 100 nm and 200 nm.
  • the drug is encapsulated in the lipidic nanoparticles in a concentration between 0.01 and 30 mg/mL.
  • the surface of the lipidic nanoparticles comprising an encapsulated drug can be modified with, but not limited to, proteins or peptides, or antibodies.
  • transferrin a protein
  • transferrin can increase the nanoparticles capacity to overcome the BBB and reach the brain.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases.
  • the present invention also relates to a method of producing the lipidic nanoparticles comprising an encapsulated drug for the treatment of neurologic or chronic diseases, comprising the following steps:
  • the temperature of the first step must be above the solid lipids melting point.
  • the lipid phase is composed of solid lipids.
  • the lipid phase is composed of a combination of solid lipids and liquid lipids wherein the solid lipids are present in a weight percentage from 0.1 to 99.9 %. The remaining quantity being liquid lipids.
  • the surfactant is used in a concentration between 1 and 10%.
  • the surfactant is selected from, but not limited to, poloxamer 407, poloxamer 188, poloxamer 182, poloxamer 908, egg lecithin, soy lecithin, polysorbate 20, polysorbate 60, or polysorbate 80.
  • the emulsion is obtained by means of a high-shear mixing device.
  • the emulsion is sonicated to reduce the size of the nanoparticles with an amplitude between 20 and 100% and a circle between 0.1 and 1.
  • the nanoparticles When the nanoparticles are left to cool down at a temperature between 20 and 30°C, it allows lipid crystallization and the nanoparticles formation.
  • the solubility of levodopa in different solid and liquid lipids was assessed through a lipid screening, in order to select the most effective lipids for SLN and NLC formulations.
  • Table 2 shows the list of lipids selected.
  • Table 2. List of lipids
  • 1 mg of this drug was mixed with 100 mg of each solid lipid.
  • the mixture of lipid/drug was heated up at 80 °C for 1 hour.
  • the solubility of the drug was determined visually, at 15-minute intervals, observing the presence or absence of crystals of the drug in the lipid.
  • Table 3 shows the results of solubility of levodopa in solid lipids.
  • NLC NLC were produced with solid and liquid lipids.
  • the previously tested levodopa-compatible lipids were then tested against each other in a proportion of 50:50 (solid lipid: liquid lipid), to evaluate their compatibility for further NLCs production.
  • the different mixtures of lipids were heated to 100°C, with stirring at 200 rpm, for 1 h. Afterwards, they were cooled to room temperature (20 ⁇ l°C), for solidification and the existence or absence of miscibility between the two lipids was then analyzed, placing a portion of each lipid mixture on filter paper, followed by visual observation, to check for the existence of drops of oil on the filter paper, which would be indicative of the lack miscibility between lipids.
  • Table 5 shows the compatibility of solid lipids and liquid lipids.
  • SLNs and NLCs were prepared by a combination of the shear homogenization method and the ultrasonication method.
  • the suspension was composed by lipids and surfactant solution.
  • NLC was produced using 350 mg of solid lipids and 150 mg of liquid lipids
  • the aqueous phase of 4.35 mL contained the surfactant poly (ethylene oxide)/poly (propylene oxide)/poly (ethylene oxide) triblock copolymer (PEO-PPO-PEO) (poloxamer 407) 10% solution;
  • the method applied consisted in: heating up the lipid and aqueous phase, separately, at a temperature between 5-10°C above the solid lipid melting point, up to melting the selected lipid; the SLN were homogenized for 0.5, 1 or 2 minutes at 12000 rpm in the Ultra-Turrax T25 (UT), followed by 5, 15 or 30 minutes of 80% intensity sonication; the NLC were homogenized for 0.5, 1 or 2 minutes at 12000 rpm in the Ultra-Turrax T25 (UT), and then sonicated during 5, 15 or 30 minutes at 80% intensity;
  • lipidic nanoparticles formulations SLN and NLC were characterized in terms of their particle size, Polydispersity Index (Pdl) and Zeta Potential values by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Instrument, UK) at 25°C. Before each measurement, the formulations were diluted in ultrapure water (1:100) to generate suitable scattering. The particle size, Pdl and Zeta Potential values of the investigated formulations were obtained by calculating the average of 3 measurements of 10 runs with 10 seconds each.
  • the chosen solid lipids for SLN formulations were the solid lipids with a decrease of levodopa crystals in the lipid screening, that were: glyceryl distearate, behenoyl polyoxyl-8 glycerides, PEG-8 beeswax, Glyceryl monostearate, Cetyl Palmitate, and glyceryl dibehenate .
  • Table 6 shows the physicochemical characterization of the SLN1 formulation glyceryl distearate + poloxamer 407.
  • Table 7 shows the physicochemical characterization of the SLN2 formulation glyceryl dibehenate + poloxamer 407.
  • Table 8 shows the physicochemical characterization of the SLN3 formulation PEG-8 beeswax + poloxamer 407.
  • Table 9 shows the physicochemical characterization of the SLN4 formulation Glyceryl Monostearate + poloxamer 407.
  • Table 10 shows the physicochemical characterization of the SLN5 formulation Cetyl Palmitate + poloxamer 407.
  • Table 11 shows the physicochemical characterization of the SLN6 formulation glyceryl dibehenate + poloxamer 407.
  • the chosen liquid lipids for NLC formulations were the liquid lipids with a decrease of levodopa crystals in the lipid screening, that were: Isopropyl myristate and medium-chain triglyceride ester of saturated coconut/palmkernel oil derived caprylic and capric fatty acids and plant derived glycerol.
  • the chosen solid lipids for NLC formulations were, according with the performance in SLN formulations: glyceryl distearate, glyceryl dibehenate and PEG-8 beeswax.
  • Table 12 shows the physicochemical characterization of the NLC1 formulation medium-chain triglycerides + glyceryl distearate + poloxamer 407.
  • Table 13 shows the physicochemical characterization of the NLC2 formulation medium-chain triglycerides + glyceryl dibehenate + poloxamer 407.
  • Table 14 shows the physicochemical characterization of the NLC3 formulation medium-chain triglycerides + PEG-8 beeswax + poloxamer 407.
  • Table 15 shows the physicochemical characterization of the NLC4 formulation Isopropyl Myristate + glyceryl distearate + poloxamer 407.
  • Table 16 shows the physicochemical characterization of the NLC5 formulation Isopropyl Myristate + glyceryl dibehenate + poloxamer 407.
  • Table 17 shows the physicochemical characterization of the NLC6 formulation Isopropyl Myristate + PEG-8 beeswax + poloxamer 407.
  • Table 12. Physicochemical characterization of the NLC1 formulation.
  • Table 18 shows the final SLN formulation parameters chosen for levodopa encapsulation.
  • Table 19 shows the final NLC formulation parameters chosen for levodopa encapsulation.
  • a stock solution was prepared dissolving levodopa in PBS (final concentration: 1.00 mg/mL), in triplicate. To produce a linear calibration curve, the stock solution was diluted with PBS to obtain the following concentration sequence: 0, 0.020, 0.040, 0.060, 0.080, 0.10 mg/mL. The absorbance of the samples was measured using the BioTek® Synergy 2 MultiMode Reader (Winooski, Vermont, USA) in a microplate reader at 280 nm. The characteristic peak of levodopa is illustrated in figure 1.
  • the equation (1) corresponds to the obtained curve of levodopa at 280 nm.
  • levodopa resistance test a thermal resistance test for the temperature above the lipid melting point to which the levodopa will be subjected during the encapsulation, once it is added to the lipid prior to its melting
  • resistance of the lipid nanoparticles production procedure test to assess whether the levodopa could degrade during the encapsulation process.
  • the absorbance spectrum of the stock solution was measured.
  • the stock solution was placed in the oven at 80 °C for 30 minutes and the absorbance spectrum was measured after cooled down at room temperature.
  • the absorbance spectrum of the stock solution was measured before and after the stock solution being placed in all steps of the lipid nanoparticles production procedure.
  • the levodopa resists to mechanic forces of the procedure equipment's to which it is subjected during encapsulation .
  • Levodopa was encapsulated into the lipidic nanoparticles at different amounts to establish the best conditions to produce stable formulations with the higher amount possible of levodopa encapsulated, and with a particle average size of less than 200 nm, ensuring the passage through BBB.
  • the prepared formulations were characterized according to their particle size, Pdl, zeta potential values, and encapsulation efficiency (EE)
  • the EE of each formulation was determined indirectly, by calculating the amount of free levodopa present in the formulations, after separation of the free drug from drug-loaded nanoparticles by a gravity separation protocol, using Sephadex G-25 columns.
  • the amount of free levodopa was quantified measuring the absorbance of free drug at the characteristic wavelength of levodopa by spectrophotometry, normalized to that of the control and the concentration of levodopa was calculated using the calibration curve obtained (equation 1).
  • Table 24 Physicochemical characterization of F5 formulation.
  • nanocarriers proved to be able to encapsulate levodopa with an encapsulation efficiency ranging from 17% to 79%. All the nanoparticles present sizes higher than 100 nm and lower than 200 nm, being suitable for brain delivery. Additionally, the nanoparticles are stable for over 6 months.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Psychology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

The present application relates to lipidic nanoparticles that are suitable for the treatment of neurologic or chronic diseases. The lipidic nanoparticles of the present application are of the solid lipid nanoparticles or nanostructured lipid carriers and comprise an encapsulated drug with a sustained and controlled drug delivery in the treatment of neurologic or chronic diseases.

Description

DESCRIPTION
"LIPIDIC NANOPARTICLES COMPRISING AN ENCAPSULATED DRUG AND METHOD OF PRODUCING THE SAME"
Technical field
This application relates to lipidic nanoparticles comprising an encapsulated drug and a method of producing the same. It also relates to a pharmaceutical composition comprising the lipidic nanoparticles.
Background art
Parkinson's Disease (PD) is the second most prevalent neurodegenerative disease, characterized by a reduction in dopamine levels in the brain, caused by a loss of nerve cells in substantia nigra, leading to movement problems once dopamine is responsible for regulating the movement of the body. The continuous increase of PD rate demands for a fast development of preventive and/or curative therapeutic strategies that are truly effective.
Levodopa is used as dopamine replacement agent for PD treatment since it is converted in dopamine when absorbed by the nerve cells in the brain, which can restore functional movements. This medication doesn't slow down or cure PD, but can control symptoms like tremors, rigidity of the muscles and bradykinesia. However, long-term treatment with levodopa is associated with physical side effects like dyskinesia and fluctuations in motor response, called on/off phenomena of PD, related with the intermittent delivery of levodopa to the brain due to drug level fluctuations in plasma. The pharmacokinetic and pharmacodynamic properties of levodopa: low diffusion through the blood brain barrier (BBB), poor bioavailability, short half-life, and conversion of levodopa in dopamine outside the brain, requires a higher drug dose to achieve the effective drug levels at the brain and/or combination of levodopa with conversion inhibitors, that leads to cardiovascular and gastrointestinal side effects.
The conventional PD therapies are available as oral pills and transdermal patches. However, long-term treatment with levodopa is associated with physical side effects like dyskinesia and fluctuations in motor response, called the wearing off of medication effect, related with the intermittent delivery of levodopa to the brain due to fluctuations of drug in plasma levels. After 5 years of levodopa intake, approximately 40% of PD patients experience motor fluctuations and/or dyskinesias. The number increases to 90% after 9 or more years of therapy with levodopa [1]. Regarding dyskinesia, this side effect is developed in about 63% and 34% of PD patients after 2 and 5 years of therapy, respectively [2,3].
Table 1. Studies of the prevalence of motor complications induced by levodopa therapy.
Figure imgf000003_0001
The development of a strategy to overcome these side-effects and improve the efficacy of the PD therapy is necessary and the use of nanoparticles (NPs) as drug carrying systems is one with a great potential.
Summary
The present invention relates to lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases, wherein the nanoparticles are solid lipid nanoparticles or are nanostructured lipid carriers composed of a mixture of solid lipids and liquid lipids.
In one embodiment the encapsulated drug is selected from levodopa, a levodopa/carbidopa combination, dopamine agonists, monoamine oxidase-B inhibitors, or amantadine.
In one embodiment the solid lipids are selected from glyceryl distearate, behenoyl polyoxyl-8 glycerides, stearoyl polyoxyl-32 glycerides, mono- or di- or triglyceride esters of fatty acids C8 to C18, glyceryl dibehenate, stearic acid, hydrogenated coco-glycerides, PEG-8 beeswax, cetyl palmitate, hydrogenated palm oil, glyceryl tristearate, glyceryl monostearate, or their mixtures.
In one embodiment the liquid lipids are selected from mediumchain triglyceride ester of saturated coconut/palmkernel oil derived caprylic and capric fatty acids and plant derived glycerol, vitamin E, decyl oleate, isopropyl myristate, medium-chain triglycerides of caprylic (C8) and capric (CIO) acids, cetyl dimeticone, or their mixtures. In one embodiment the solid lipids are present in a weight percentage from 0.1 to 99.9 % in the mixture of solid lipids and liquid lipids, the remaining quantity being liquid lipids.
In one embodiment the nanoparticles comprise an average size between 100 nm and 200 nm.
In one embodiment the drug is encapsulated in the lipidic nanoparticles in a concentration between 0.01 and 30 mg/mL.
In one embodiment the surface of the lipidic nanoparticles is modified with proteins or peptide, or antibodies.
In one embodiment the neurologic or chronic diseases are selected from epilepsy, Alzheimer's and other dementias, strokes, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, neurological infections, brain tumors, major depression, schizophrenia, attention deficit hyperactivity disorder or Autism.
The present invention also relates to a pharmaceutical composition comprising the lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases.
The present invention also relates to a method of producing the lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases, comprising the following steps:
Heating a lipid phase of solid lipids or a lipid phase of a solid/liquid lipids combination, with a drug to a temperature 5 to 10°C above the melting point of the solid lipids;
Heating an aqueous phase comprising a surfactant to the same temperature used in the previous step;
Disperse the lipid phase in the aqueous phase by mixing them;
Obtaining an emulsion by homogenizing the previous mixture for a time between 30 seconds and 2 minutes;
Sonicating the emulsion for a time between 1 and 30 minutes;
Cool down the emulsion to a temperature between 20 and 30 °C.
In one embodiment the surfactant is selected from poloxamer 407, poloxamer 188, poloxamer 182, poloxamer 908, egg lecithin, soy lecithin, polysorbate 20, polysorbate 60, or polysorbate 80.
In one embodiment the surfactant is used in a concentration between 1 and 10%.
General Description
The present invention relates to biocompatible and biodegradable lipidic nanoparticles (NPs) comprising an encapsulated drug for the treatment of a neurologic or a chronic disease, for example levodopa, that will provide a sustained and controlled release of said drug to increase the therapeutic effect of the drug and reduce side effects.
For example, in neurologic diseases in the brain, the drug will be released from the NPs and will achieve effective drug levels at the required location, decreasing the side effects for the other tissues/organs. Since the selected NPs have optimal clearance characteristics, the NPs will be degraded after releasing their load, reducing potential side effects due to NPs toxicity.
As the drug will be released over time, achieving a continuous drug delivery, patients will consistently receive their treatment without interruptions, reducing the on/off phenomena of neurologic diseases such as PD. Although with NPs as carriers for drugs for the treatment of PD, only a low amount of the drug is required to achieve the same effect as the ones that are currently being used for the PD patients as patches or pills.
These nanoparticles have potential to be used to encapsulate different active molecules and can be applied for the treatment of other neurological or chronic diseases.
The presently disclosed lipidic nanoparticles present sizes lower than 200 nm, stability over several months (6 months) and encapsulation efficiencies up to 79%.
Compared to the previously reported nanoparticles encapsulating levodopa (poly (lactic-co-glycolic acid) (PLGA) nanoparticles and liposomes), the present invention presents several advantages. These PLGA nanoparticles and liposomes present several inadequate properties to be used in the treatment of PD, such as large size (above 200 nm) and/or low stability and/or lower encapsulation efficiencies [7 - 11]. Therefore, the present invention represents a novel approach with improved features to treat neurologic or chronic diseases .
Neurologic diseases and chronic diseases can be selected from, but not limited to, epilepsy, Alzheimer's and other dementias, strokes, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, neurological infections, brain tumors, major depression, schizophrenia, attention deficit hyperactivity disorder, Autism.
The performance of a PD therapy using a drug such as levodopa encapsulated into the lipidic NPs will be increased, since the sustained and continuous release of a drug from NPs will maintain the active doses of drug, reducing the wearing off of medication effect, making therapy truly effective. Since the NPs have ability to reach the brain, there will be a significant increase of the drug at the targeted organ (brain), providing reduced systemic toxicity with less side effects, and lower doses will be required, compared to the conventional therapy.
For the purposes of this application, a drug is understood as an active molecule intended for use in preventing or curing a disease or relieving pain or disease symptoms.
As proof of concept, levodopa-loaded NPs were successfully produced with higher EE, using solid lipid nanoparticles (NPLs) and nanostructured lipid carriers (NLCs) as carriers. The successfully loaded-levodopa formulations showed a size smaller than 200 nm, an ideal size for systemic administration and passing the BBB with a narrow-size distribution. Regarding the ZP values, levodopa encapsulation did not significantly impact the surface charge of the NPs. The ZP values of the formulation were high enough to ensure their stability after systemic administration, avoiding NPs aggregation. The levodopa- loaded SLNs and levodopa-loaded NLCs remained stable and did not form aggregates over 6 months.
Brief description of drawings
For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.
Figure 1 shows the structure of levodopa.
Figure 2 shows a UV-Vis Spectrum obtained for levodopa with indication of the characteristic absorbance peaks of this drug at 0.1 mg/ml (280 nm).
Figure 3 shows calibration curve of levodopa in wavelength 280 nm (mean ± SD, n = 3).
Figure 4 shows the results of temperature resistance test of levodopa (mean ± SD, n = 3).
Figure 5 shows the results of lipid nanoparticles production procedure resistance test of levodopa (mean ± SD, n = 3).
Figure 6 shows the results of levodopa stability study with temperature at 4°C and 25°C (mean ± SD, n = 3). Detailed description of embodiments
Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.
The present invention relates to lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases. The nanoparticles of the present invention are of the solid lipid nanoparticles (SLN) type - composed of solid lipids - or nanostructured lipid carriers (NLC) type - composed of a mixture of solid lipids and liquid lipids.
The encapsulated drug is selected from, but not limited to, levodopa, a levodopa/carbidopa combination, dopamine agonists, monoamine oxidase-B inhibitors, or amantadine.
In one embodiment the nanoparticles comprise solid lipids selected from, but not limited to, glyceryl distearate, behenoyl polyoxyl-8 glycerides, stearoyl polyoxyl-32 glycerides, mono- or di- or triglyceride esters of fatty acids C8 to C18, glyceryl dibehenate, stearic acid, hydrogenated coco-glycerides, PEG-8 beeswax, cetyl palmitate, hydrogenated palm oil, glyceryl tristearate, glyceryl monostearate, or their mixtures.
In one embodiment, in the mixture of solid lipids and liquid lipids, the solid lipids are present in a weight percentage from 0.1 to 99.9 %. The remaining quantity being liquid lipids. In one embodiment the nanoparticles comprise solid lipids selected from, but not limited to, glyceryl distearate, behenoyl polyoxyl-8 glycerides, stearoyl polyoxyl-32 glycerides, mono- or di- or triglyceride esters of fatty acids (C8 to C18), glyceryl dibehenate, stearic acid, hydrogenated coco-glycerides, PEG-8 beeswax, cetyl palmitate, hydrogenated palm oil, glyceryl tristearate, glyceryl monostearate, or their mixtures.
In one embodiment the liquid lipids are selected from, but not limited to, medium-chain triglyceride ester of saturated coconut/palmkernel oil derived caprylic and capric fatty acids and plant derived glycerol, vitamin E, decyl oleate, isopropyl myristate, medium-chain triglycerides of caprylic (C8) and capric (CIO) acids, cetyl dimeticone, or their mixtures.
In one embodiment, the nanoparticles comprise an average size between 100 nm and 200 nm.
In one embodiment, the drug is encapsulated in the lipidic nanoparticles in a concentration between 0.01 and 30 mg/mL.
In one embodiment, the surface of the lipidic nanoparticles comprising an encapsulated drug can be modified with, but not limited to, proteins or peptides, or antibodies.
For example, transferrin (a protein) can increase the nanoparticles capacity to overcome the BBB and reach the brain.
The present invention also relates to a pharmaceutical composition comprising the lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases.
The present invention also relates to a method of producing the lipidic nanoparticles comprising an encapsulated drug for the treatment of neurologic or chronic diseases, comprising the following steps:
- Heating a lipid phase of solid lipids or a lipid phase of a solid/liquid lipids combination, with a drug to a temperature 5 to 10°C above the melting point of the solid lipids;
- Heating an aqueous phase comprising a surfactant to the same temperature used in the previous step;
- Disperse the lipid phase in the aqueous phase by mixing them;
- Obtaining an emulsion by homogenizing the previous mixture for a time between 30 seconds and 2 minutes;
- Sonicating the emulsion for a time between 1 and 30 minutes;
- Cool down the emulsion to a temperature between 20 and 30 °C.
The temperature of the first step must be above the solid lipids melting point.
In the embodiment of the SLNs, the lipid phase is composed of solid lipids.
In the embodiment of the NLCs, the lipid phase is composed of a combination of solid lipids and liquid lipids wherein the solid lipids are present in a weight percentage from 0.1 to 99.9 %. The remaining quantity being liquid lipids.
In one embodiment, the surfactant is used in a concentration between 1 and 10%. In one embodiment, the surfactant is selected from, but not limited to, poloxamer 407, poloxamer 188, poloxamer 182, poloxamer 908, egg lecithin, soy lecithin, polysorbate 20, polysorbate 60, or polysorbate 80.
In one embodiment the emulsion is obtained by means of a high-shear mixing device.
The emulsion is sonicated to reduce the size of the nanoparticles with an amplitude between 20 and 100% and a circle between 0.1 and 1.
When the nanoparticles are left to cool down at a temperature between 20 and 30°C, it allows lipid crystallization and the nanoparticles formation.
Proof of concept was performed selecting levodopa as drug to be encapsulated in the lipidic nanoparticles, and the results are disclosed below.
Examples
1. Development and characterization of lipidic nanoparticles (SLN and NLC)
1.1 Screening of solid and liquid lipids
The solubility of levodopa in different solid and liquid lipids was assessed through a lipid screening, in order to select the most effective lipids for SLN and NLC formulations. Table 2 shows the list of lipids selected. Table 2. List of lipids
Figure imgf000014_0001
For the study of levodopa compatibility/solubility in solid lipids, 1 mg of this drug was mixed with 100 mg of each solid lipid. The mixture of lipid/drug was heated up at 80 °C for 1 hour. The solubility of the drug was determined visually, at 15-minute intervals, observing the presence or absence of crystals of the drug in the lipid. Table 3 shows the results of solubility of levodopa in solid lipids.
Table 3. Solubility of levodopa in solid lipids.
Figure imgf000015_0001
Figure imgf000016_0001
(+) presence of crystals of the drug; (-) absence of crystals of the drug; (*) with a decrease of crystals of the drug.
For the compatibility/solubility study of levodopa in liquid lipids, 1 mg of the drug was mixed with 100 mg of each liquid lipid. The mixture of lipid/drug was heated up at 80 °C for 1 hour with vigorous stirring, followed by visual observation to verify the presence or absence of crystals of the drug. Table 4 shows the results of the solubility of levodopa in liquid lipids.
Table 4. Solubility of levodopa in liquid lipids.
Figure imgf000016_0002
Figure imgf000017_0002
(+) presence of crystals of the drug; (-) absence of crystals of the drug; (*) with a decrease of crystals of the drug.
NLC were produced with solid and liquid lipids. The previously tested levodopa-compatible lipids were then tested against each other in a proportion of 50:50 (solid lipid: liquid lipid), to evaluate their compatibility for further NLCs production. The different mixtures of lipids were heated to 100°C, with stirring at 200 rpm, for 1 h. Afterwards, they were cooled to room temperature (20±l°C), for solidification and the existence or absence of miscibility between the two lipids was then analyzed, placing a portion of each lipid mixture on filter paper, followed by visual observation, to check for the existence of drops of oil on the filter paper, which would be indicative of the lack miscibility between lipids. Table 5 shows the compatibility of solid lipids and liquid lipids.
Table 5. Compatibility of solid lipids and liquid lipids.
Figure imgf000017_0001
Figure imgf000018_0001
1.2 Optimization of lipidic nanoparticle's formulations
The optimization of SLN and NLC in terms of physicochemical characteristics was performed according with the lipids previously chosen, to develop suitable formulations to encapsulate levodopa able to cross the blood-brain barrier.
Nanoparticle's preparation:
SLNs and NLCs were prepared by a combination of the shear homogenization method and the ultrasonication method. The suspension was composed by lipids and surfactant solution.
• SLN was produced using 500 mg of the solid lipids;
• NLC was produced using 350 mg of solid lipids and 150 mg of liquid lipids;
• The aqueous phase of 4.35 mL contained the surfactant poly (ethylene oxide)/poly (propylene oxide)/poly (ethylene oxide) triblock copolymer (PEO-PPO-PEO) (poloxamer 407) 10% solution;
The method applied consisted in: heating up the lipid and aqueous phase, separately, at a temperature between 5-10°C above the solid lipid melting point, up to melting the selected lipid; the SLN were homogenized for 0.5, 1 or 2 minutes at 12000 rpm in the Ultra-Turrax T25 (UT), followed by 5, 15 or 30 minutes of 80% intensity sonication; the NLC were homogenized for 0.5, 1 or 2 minutes at 12000 rpm in the Ultra-Turrax T25 (UT), and then sonicated during 5, 15 or 30 minutes at 80% intensity;
Cooling down at a temperature between 20 and 25 °C, allowing the lipid crystallization and the nanoparticles formation.
1.2.1 Characterization of lipidic nanoparticles formulations SLN and NLC were characterized in terms of their particle size, Polydispersity Index (Pdl) and Zeta Potential values by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Instrument, UK) at 25°C. Before each measurement, the formulations were diluted in ultrapure water (1:100) to generate suitable scattering. The particle size, Pdl and Zeta Potential values of the investigated formulations were obtained by calculating the average of 3 measurements of 10 runs with 10 seconds each.
1.2.1.1 SLN formulations
The chosen solid lipids for SLN formulations were the solid lipids with a decrease of levodopa crystals in the lipid screening, that were: glyceryl distearate, behenoyl polyoxyl-8 glycerides, PEG-8 beeswax, Glyceryl monostearate, Cetyl Palmitate, and glyceryl dibehenate .
Table 6 shows the physicochemical characterization of the SLN1 formulation glyceryl distearate + poloxamer 407. Table 7 shows the physicochemical characterization of the SLN2 formulation glyceryl dibehenate + poloxamer 407. Table 8 shows the physicochemical characterization of the SLN3 formulation PEG-8 beeswax + poloxamer 407. Table 9 shows the physicochemical characterization of the SLN4 formulation Glyceryl Monostearate + poloxamer 407. Table 10 shows the physicochemical characterization of the SLN5 formulation Cetyl Palmitate + poloxamer 407. Table 11 shows the physicochemical characterization of the SLN6 formulation glyceryl dibehenate + poloxamer 407.
Table 6. Physicochemical characterization of the SLN1 formulation .
Figure imgf000020_0001
Figure imgf000021_0001
Table 7. Physicochemical characterization of the SLN2 formulation .
Figure imgf000021_0002
Table 8. Physicochemical characterization of the SLN3 formulation .
Figure imgf000022_0001
Table 9. Physicochemical characterization of the SLN4 formulation .
Figure imgf000022_0002
Figure imgf000023_0001
Table 10. Physicochemical characterization of the SLN5 formulation.
Figure imgf000023_0002
Figure imgf000024_0001
Table 11. Physicochemical characterization of the SLN6 formulation.
Figure imgf000024_0002
Figure imgf000025_0001
1.2.1.2 NLC formulations
The chosen liquid lipids for NLC formulations were the liquid lipids with a decrease of levodopa crystals in the lipid screening, that were: Isopropyl myristate and medium-chain triglyceride ester of saturated coconut/palmkernel oil derived caprylic and capric fatty acids and plant derived glycerol. The chosen solid lipids for NLC formulations were, according with the performance in SLN formulations: glyceryl distearate, glyceryl dibehenate and PEG-8 beeswax.
Table 12 shows the physicochemical characterization of the NLC1 formulation medium-chain triglycerides + glyceryl distearate + poloxamer 407. Table 13 shows the physicochemical characterization of the NLC2 formulation medium-chain triglycerides + glyceryl dibehenate + poloxamer 407. Table 14 shows the physicochemical characterization of the NLC3 formulation medium-chain triglycerides + PEG-8 beeswax + poloxamer 407. Table 15 shows the physicochemical characterization of the NLC4 formulation Isopropyl Myristate + glyceryl distearate + poloxamer 407. Table 16 shows the physicochemical characterization of the NLC5 formulation Isopropyl Myristate + glyceryl dibehenate + poloxamer 407. Table 17 shows the physicochemical characterization of the NLC6 formulation Isopropyl Myristate + PEG-8 beeswax + poloxamer 407. Table 12. Physicochemical characterization of the NLC1 formulation.
Figure imgf000026_0001
Table 13. Physicochemical characterization of the NLC2 formulation.
Figure imgf000026_0002
Figure imgf000027_0001
Table 14. Physicochemical characterization of the NLC3 formulation.
Figure imgf000027_0002
Figure imgf000028_0001
Table 15. Physicochemical characterization of the NLC4 formulation .
Figure imgf000028_0002
Figure imgf000029_0001
Table 16. Physicochemical characterization of the NLC5 formulation.
Figure imgf000029_0002
Table 17. Physicochemical characterization of the NLC6 formulation.
Figure imgf000030_0001
Table 18 shows the final SLN formulation parameters chosen for levodopa encapsulation.
Table 18. SLN formulation parameters.
Figure imgf000030_0002
Figure imgf000031_0001
Table 19 shows the final NLC formulation parameters chosen for levodopa encapsulation.
Table 19. NLC formulation parameters.
Figure imgf000031_0002
2. Levodopa
2.1 Calibration curve
For quantification purposes, the calibration curve of levodopa was generated.
A stock solution was prepared dissolving levodopa in PBS (final concentration: 1.00 mg/mL), in triplicate. To produce a linear calibration curve, the stock solution was diluted with PBS to obtain the following concentration sequence: 0, 0.020, 0.040, 0.060, 0.080, 0.10 mg/mL. The absorbance of the samples was measured using the BioTek® Synergy 2 MultiMode Reader (Winooski, Vermont, USA) in a microplate reader at 280 nm. The characteristic peak of levodopa is illustrated in figure 1.
To produce a linear calibration curve of levodopa in PBS, solutions with concentrations known were prepared, the absorbance of the samples was measured, and calibration curves were obtained (figure 2).
The equation (1) corresponds to the obtained curve of levodopa at 280 nm.
Abs = 8.5079 C - 0.0119 (1)
R2 = 0.9977
The results demonstrate a linear relationship between concentration and absorbance, confirmed by the slope of the linear calibration curve. The obtained calibration curve has a high coefficient of determination (R2), which translates into an adequate linear relationship between concentration and absorbance. 2.2 Levodopa resistance analysis
Two tests of levodopa resistance were performed: temperature resistance test (a thermal resistance test for the temperature above the lipid melting point to which the levodopa will be subjected during the encapsulation, once it is added to the lipid prior to its melting) and resistance of the lipid nanoparticles production procedure test, to assess whether the levodopa could degrade during the encapsulation process. Stock solutions with the following concentrations: 0, 0.020, 0.040, 0.060, 0.080, 0.10 mg/mL, were prepared in ultrapure water in order to study the levodopa behaviour at these concentrations.
For the temperature resistance test, the absorbance spectrum of the stock solution was measured. The stock solution was placed in the oven at 80 °C for 30 minutes and the absorbance spectrum was measured after cooled down at room temperature.
Analyzing the graph of figure 4, it is possible to observe that there was no degradation of the levodopa, since their absorbance values after exposure to the temperature of 80°C remains the same as the values for 25°C. Thus, the levodopa resist to the melting temperature of the lipid to which it is subjected during encapsulation, has the melting point of the levodopa is 276-278 °C.
For the resistance to the lipid nanoparticles production procedure, the absorbance spectrum of the stock solution was measured before and after the stock solution being placed in all steps of the lipid nanoparticles production procedure.
Analysing figure 4, it is possible to verify that there was no degradation of levodopa, since the absorbance values, in the study concentrations, after the lipid nanoparticles production procedure remains the same as the ones obtained before starting the procedure.
Thus, the levodopa resists to mechanic forces of the procedure equipment's to which it is subjected during encapsulation .
2.3 Levodopa stability
The loss of drugs health-promoting effects is very common to occur, once they are susceptible to degradation, not only in biological environment, but also during their processing and storage. The stability study with temperature was conducted in order to understand the best storage conditions for levodopa, and their ideal period of storage.
In order to study the levodopa stability with temperature, stock solutions were prepared dissolving the compound in ultrapure water (final concentration: 0.10 mg/mL) and stored at two different temperatures: 4°C and 25°C, to test the storage conditions, for a period of 30 days, protected from light. At predicted days, the absorbance of the samples was measured using BioTek® Synergy 2 Multi-Mode Reader (Winooski, Vermont, USA) in a microplate reader at the characteristic wavelength of levodopa.
The results of stability with temperature (figure 5) demonstrated that levodopa is stable and with no sign of degradation for 30 days when stored at 4°C and 25°C, once there are no statistically significant alterations in their absorbance values over time.
All tests and trials were performed in triplicate and the average of the obtained absorbances was calculated, normalized to that of the control (Abs compound - Abs control) and plotted against concentration to establish the graph.
3. Levodopa-loaded lipidic nanoparticles
Levodopa was encapsulated into the lipidic nanoparticles at different amounts to establish the best conditions to produce stable formulations with the higher amount possible of levodopa encapsulated, and with a particle average size of less than 200 nm, ensuring the passage through BBB.
The prepared formulations were characterized according to their particle size, Pdl, zeta potential values, and encapsulation efficiency (EE) The EE of each formulation was determined indirectly, by calculating the amount of free levodopa present in the formulations, after separation of the free drug from drug-loaded nanoparticles by a gravity separation protocol, using Sephadex G-25 columns. The amount of free levodopa was quantified measuring the absorbance of free drug at the characteristic wavelength of levodopa by spectrophotometry, normalized to that of the control and the concentration of levodopa was calculated using the calibration curve obtained (equation 1).
The difference between the amount used in the formulation synthesis and the amount that remained free in the aqueous phase, allow us to determine the EE, as described at equation 2, as follows:
Figure imgf000035_0001
3.1 Solid lipid Nanoparticles (SLN)
Table 20 shows the physicochemical characterization of Fl formulation glyceryl distearate + poloxamer 407 (mean ± SD, n = 3).
Table 20. physicochemical characterization of Fl formulation.
Figure imgf000036_0001
Table 21 shows the physicochemical characterization of F2 formulation behenoyl polyoxyl-8 glycerides + poloxamer 407 (mean ± SD, n = 3).
Table 21. Physicochemical characterization of F2.
Figure imgf000036_0002
Figure imgf000037_0001
Table 22 shows the physicochemical characterization of F3 formulation PEG-8 beeswax + poloxamer 407 (mean ± SD, n = 3).
Table 22. Physicochemical characterization of F3.
Figure imgf000037_0002
3.2 Nanostructured Lipid Carriers (NLC)
Table 23 shows the physicochemical characterization of F4 formulation Isopropyl myristate + glyceryl distearate + poloxamer 407 (mean ± SD, n = 3).
Table 23. Physicochemical characterization of F4 formulation .
Figure imgf000038_0002
Table 24 shows the physicochemical characterization of F5 formulation Isopropyl myristate + behenoyl polyoxyl-8 glycerides + poloxamer 407 (mean ± SD, n = 3).
Table 24. Physicochemical characterization of F5 formulation.
Figure imgf000038_0001
Table 25 shows the physicochemical characterization of F6 formulation Isopropyl myristate + PEG-8 beeswax + poloxamer 407 (mean ± SD, n = 3).
Table 25. Physicochemical characterization of F6 formulation.
Figure imgf000039_0001
Table 26 shows the physicochemical characterization of F7 formulation medium-chain triglycerides + glyceryl distearate + poloxamer 407 (mean ± SD, n = 3).
Table 26. Physicochemical characterization of F7 formulation.
Figure imgf000039_0002
Figure imgf000040_0001
Table 27. Physicochemical characterization of F8 formulation.
Figure imgf000040_0002
Figure imgf000041_0001
Conclusions:
Different formulations containing SLN and NLC were produced using different materials and conditions. Those nanocarriers proved to be able to encapsulate levodopa with an encapsulation efficiency ranging from 17% to 79%. All the nanoparticles present sizes higher than 100 nm and lower than 200 nm, being suitable for brain delivery. Additionally, the nanoparticles are stable for over 6 months.
This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims. The preferred forms of implementation described above can obviously be combined with each other. The following claims further define the preferred forms of implementation.
[1] Ahlskog JE, Muenter MD. Frequency of levopdopa-related dyskinesias and motor fluctuations as estimated from the cumulative literature. Mov Disord. 2001;16(3):448-458.
[2] Holloway RG, Shoulson I, Fahn S, et al. Pramipexole vs levodopa as initial treatment for Parkinson's disease: a 4- year randomized, controlled trial. Arch Neurol. 2004;61:1044-1053.
[3] Rascol 0, Brooks DJ, Korczyn AD, et al. A five-year study of the incidence of dyskinesia in patients with early Parkinson's disease who were treated with ropinirole or levodopa. N Engl J Med. 2000;342:1484-1491. [4] Schrag A, Quinn N (2000) Dyskinesias and motor fluctuations in Parkinson's disease. A community-based study. Brain 123(Pt 11):2297-305
[5] Scott NW, Macleod AD, Counsell CE (2016) Motor complications in an incident Parkinson's disease cohort. Eur J Neurol 232(2):304-312
[6] Bjornestad A, Forsaa EB, Pedersen KF, Tysnes OB, Larsen JP, Alves G (2016) Risk and course of major complications in a population-based incident Parkinson's disease cohort. Parkinsonism Relat Disord 22:48-53
[7] Moholkar, D. N.; Sadalage, P. S.; Havaldar, D. V.; Pawar, K. D., Engineering the liposomal formulations from natural peanut phospholipids for pH and temperature sensitive release of folic acid, levodopa and camptothecin. Mater Sci Eng C Mater Biol Appl 2021, 123, 111979.
[8] Gurturk, Z.; Tezcaner, A.; Dalgic, A. D.; Korkmaz, S.; Keskin, D., Maltodextrin modified liposomes for drug delivery through the b lood-brain barrier. Medchemcomm 2017, 8 (6), 1337-1345.
[9] Garcia Esteban, E.; Cozar-Bernal, M. J.; Rabasco Alvarez, A. M.; Gonzalez-Rodriguez, M. L., A comparative study of stabilising effect and antioxidant activity of different antioxidants on levodopa-loaded liposomes. J Microencapsul 2018, 35 (4), 357-371.
[10] Zhou, Y. Z.; Alany, R. G.; Chuang, V.; Wen, J., Optimization of PLGA nanoparticles formulation containing L- DOPA by applying the central composite design. Drug Dev Ind Pharm 2013, 39 (2), 321-30.
[11] Arisoy, S.; Sayiner, 0.; Comoglu, T.; Onal, D.; Atalay, 0.; Pehlivanoglu, B., In vitro and in vivo evaluation of levodopa-loaded nanoparticles for nose to brain delivery. Pharm Dev Technol 2020, 25 (6), 735-747.)

Claims

1. Lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases, wherein the nanoparticles are solid lipid nanoparticles or are nanostructured lipid carriers composed of a mixture of solid lipids and liquid lipids.
2. Lipidic nanoparticles according to the previous claim, wherein the encapsulated drug is selected from levodopa, a levodopa/carbidopa combination, dopamine agonists, monoamine oxidase-B inhibitors, or amantadine.
3. Lipidic nanoparticles according to any of the previous claims, wherein the solid lipids are selected from glyceryl distearate, behenoyl polyoxyl-8 glycerides, stearoyl polyoxyl-32 glycerides, mono- or di- or triglyceride esters of fatty acids C8 to C18, glyceryl dibehenate, stearic acid, hydrogenated coco-glycerides, PEG-8 beeswax, cetyl palmitate, hydrogenated palm oil, glyceryl tristearate, glyceryl monostearate, or their mixtures.
4. Lipidic nanoparticles according to any of the previous claims, wherein the liquid lipids are selected from mediumchain triglyceride ester of saturated coconut/palmkernel oil derived caprylic and capric fatty acids and plant derived glycerol, vitamin E, decyl oleate, isopropyl myristate, medium-chain triglycerides of caprylic (C8) and capric (CIO) acids, cetyl dimeticone, or their mixtures.
5. Lipidic nanoparticles according to any of the previous claims, wherein the solid lipids are present in a weight percentage from 0.1 to 99.9 % in the mixture of solid lipids and liquid lipids, the remaining quantity being liquid lipids.
6. Lipidic nanoparticles according to any of the previous claims, wherein the nanoparticles comprise an average size between 100 nm and 200 nm.
7. Lipidic nanoparticles according to any of the previous claims, wherein the drug is encapsulated in the lipidic nanoparticles in a concentration between 0.01 and 30 mg/mL.
8. Lipidic nanoparticles according to any of the previous claims, wherein the surface of the lipidic nanoparticles is modified with proteins or peptide, or antibodies.
9. Lipidic nanoparticles according to any of the previous claims, wherein the neurologic or chronic diseases are selected from epilepsy, Alzheimer's and other dementias, strokes, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, neurological infections, brain tumors, major depression, schizophrenia, attention deficit hyperactivity disorder or Autism.
10. A pharmaceutical composition comprising the lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases described in any of the claims 1 to 9.
11. Method of producing the lipidic nanoparticles comprising an encapsulated drug suitable for the treatment of neurologic or chronic diseases described in any of the claims 1 to 9, characterized by comprising the following steps: Heating a lipid phase of solid lipids or a lipid phase of a solid/liquid lipids combination, with a drug to a temperature 5 to 10°C above the melting point of the solid lipids;
Heating an aqueous phase comprising a surfactant to the same temperature used in the previous step;
Disperse the lipid phase in the aqueous phase by mixing them;
Obtaining an emulsion by homogenizing the previous mixture for a time between 30 seconds and 2 minutes;
Sonicating the emulsion for a time between 1 and 30 minutes;
Cool down the emulsion to a temperature between 20 and 30 °C.
12. Method according to the previous claim, wherein the surfactant is selected from poloxamer 407, poloxamer 188, poloxamer 182, poloxamer 908, egg lecithin, soy lecithin, polysorbate 20, polysorbate 60, or polysorbate 80.
13. Method according to any of the claim 11 to 12, wherein the surfactant is used in a concentration between 1 and 10%.
PCT/IB2023/060950 2022-10-31 2023-10-31 Lipidic nanoparticles comprising an encapsulated drug and method of producing the same WO2024095144A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PT11829722 2022-10-31
PT118297 2022-10-31

Publications (1)

Publication Number Publication Date
WO2024095144A1 true WO2024095144A1 (en) 2024-05-10

Family

ID=88969549

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2023/060950 WO2024095144A1 (en) 2022-10-31 2023-10-31 Lipidic nanoparticles comprising an encapsulated drug and method of producing the same

Country Status (1)

Country Link
WO (1) WO2024095144A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130017239A1 (en) * 2010-03-24 2013-01-17 Lipotec S.A. Lipid nanoparticle capsules

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130017239A1 (en) * 2010-03-24 2013-01-17 Lipotec S.A. Lipid nanoparticle capsules

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
AGRAWAL MUKTA ET AL: "Recent strategies and advances in the fabrication of nano lipid carriers and their application towards brain targeting", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 321, 12 February 2020 (2020-02-12), pages 372 - 415, XP086135623, ISSN: 0168-3659, [retrieved on 20200212], DOI: 10.1016/J.JCONREL.2020.02.020 *
AHLSKOG JEMUENTER MD: "Frequency of levopdopa-related dyskinesias and motor fluctuations as estimated from the cumulative literature", MOV DISORD, vol. 16, no. 3, 2001, pages 448 - 458
ARISOY, S.SAYINER, O.COMOGLU, T.ONAL, D.ATALAY, 0.: "Pehlivanoglu, B., In vitro and in vivo evaluation of levodopa-loaded nanoparticles for nose to brain delivery", PHARM DEV TECHNOL, vol. 25, no. 6, 2020, pages 735 - 747
BJORNESTAD AFORSAA EBPEDERSEN KFTYSNES OBLARSEN JPALVES G: "Risk and course of major complications in a population-based incident Parkinson's disease cohort", PARKINSONISM RELAT DISORD, vol. 22, 2016, pages 48 - 53, XP029362544, DOI: 10.1016/j.parkreldis.2015.11.007
DATABASE CAPLUS [online] STNext; 8 November 2010 (2010-11-08), ZHAN SHUIMING ET AL: "Preparation and entrapment efficiency determination of solid lipid nanoparticles loaded levodopa", XP093120865, Database accession no. 2010:1382375 *
GARCIA ESTEBAN, E.COZAR-BERNAL, M. J.RABASCO ALVAREZ, A. M.GONZALEZ-RODRIGUEZ, M. L.: "A comparative study of stabilising effect and antioxidant activity of different antioxidants on levodopa-loaded liposomes", J MICROENCAPSUL, vol. 35, no. 4, 2018, pages 357 - 371
GURTURK, Z.TEZCANER, A.DALGIC, A. D.KORKMAZ, S.KESKIN, D.: "Maltodextrin modified liposomes for drug delivery through the b lood-brain barrier", MEDCHEMCOMM, vol. 8, no. 6, 2017, pages 1337 - 1345
HOLLOWAY RGSHOULSON IFAHN S ET AL.: "ramipexole vs levodopa as initial treatment for Parkinson's disease: a 4-year randomized, controlled trial", ARCH NEUROL, vol. 61, 2004, pages 1044 - 1053
IVANA CACCIATORE ET AL: "Solid lipid nanoparticles as a drug delivery system for the treatment of neurodegenerative diseases", EXPERT OPINION ON DRUG DELIVERY, vol. 13, no. 8, 2 August 2016 (2016-08-02), GB, pages 1121 - 1131, XP055768252, ISSN: 1742-5247, DOI: 10.1080/17425247.2016.1178237 *
KARTHIVASHAN GOVINDARAJAN ET AL: "Lipid-based nanodelivery approaches for dopamine-replacement therapies in Parkinson's disease: From preclinical to translational studies", BIOMATERIALS, ELSEVIER, AMSTERDAM, NL, vol. 232, 23 December 2019 (2019-12-23), XP086001939, ISSN: 0142-9612, [retrieved on 20191223], DOI: 10.1016/J.BIOMATERIALS.2019.119704 *
MOHOLKAR, D. N.SADALAGE, P. S.HAVALDAR, D. V.PAWAR, K. D.: "Engineering the liposomal formulations from natural peanut phospholipids for pH and temperature sensitive release of folic acid, levodopa and camptothecin", MATER SCI ENG C MATER BIOL APPL, vol. 123, 2021, pages 111979
RASCOL 0BROOKS DJKORCZYN AD ET AL.: "A five-year study of the incidence of dyskinesia in patients with early Parkinson's disease who were treated with ropinirole or levodopa", N ENGL J MED, vol. 342, 2000, pages 1484 - 1491
SCHRAG AQUINN N: "Dyskinesias and motor fluctuations in Parkinson's disease. A community-based study", BRAIN, vol. 123, 2000, pages 2297 - 305
SCOTT NWMACLEOD ADCOUNSELL CE: "Motor complications in an incident Parkinson's disease cohort", EUR J NEUROL, vol. 232, no. 2, 2016, pages 304 - 312
TAPEINOS CHRISTOS ET AL: "Advances in the design of solid lipid nanoparticles and nanostructured lipid carriers for targeting brain diseases", JOURNAL OF CONTROLLED RELEASE, vol. 264, 1 October 2017 (2017-10-01), AMSTERDAM, NL, pages 306 - 332, XP093120735, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2017.08.033 *
TEIXEIRA M I ET AL: "Current insights on lipid nanocarrier-assisted drug delivery in the treatment of neurodegenerative diseases", EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, ELSEVIER SCIENCE PUBLISHERS B.V., AMSTERDAM, NL, vol. 149, 23 January 2020 (2020-01-23), pages 192 - 217, XP086081900, ISSN: 0939-6411, [retrieved on 20200123], DOI: 10.1016/J.EJPB.2020.01.005 *
WITIKA BWALYA ANGEL ET AL: "Lipid-Based Nanocarriers for Neurological Disorders: A Review of the State-of-the-Art and Therapeutic Success to Date", PHARMACEUTICS, vol. 14, no. 4, 11 April 2022 (2022-04-11), CH, pages 836, XP093120539, ISSN: 1999-4923, DOI: 10.3390/pharmaceutics14040836 *
ZHAN SHUIMING ET AL: "Preparation and entrapment efficiency determination of solid lipid nanoparticles loaded levodopa", ZHONGGUO YIYUAN YAOXUE ZAZHI BIANJIBU (CHIN. J. HOSP. PHARM ), vol. 14, 1 January 2010 (2010-01-01), pages 1171 - 1175, XP093120867 *
ZHOU, Y. Z.ALANY, R. G.CHUANG, V.WEN, J.: "Optimization of PLGA nanoparticles formulation containing L-DOPA by applying the central composite design", DRUG DEV IND PHARM, vol. 39, no. 2, 2013, pages 321 - 30

Similar Documents

Publication Publication Date Title
US20220304938A1 (en) Lipid nanoparticle compositions and methods as carriers of cannabinoids in standardized precision-metered dosage forms
Tsai et al. Baicalein loaded in tocol nanostructured lipid carriers (tocol NLCs) for enhanced stability and brain targeting
Abbas et al. Superparamagnetic iron oxide–loaded lipid nanocarriers incorporated in thermosensitive in situ gel for magnetic brain targeting of clonazepam
Tsai et al. Oral apomorphine delivery from solid lipid nanoparticles with different monostearate emulsifiers: pharmacokinetic and behavioral evaluations
Fang et al. Nanostructured lipid carriers (NLCs) for drug delivery and targeting
Esposito et al. Nanoparticulate lipid dispersions for bromocriptine delivery: Characterization and in vivo study
Sastri et al. Solid lipid nanoparticles: Preparation techniques, their characterization, and an update on recent studies
Zhou et al. Nanostructured lipid carriers used for oral delivery of oridonin: an effect of ligand modification on absorption
Zhang et al. Enhancement of oral bioavailability of tripterine through lipid nanospheres: preparation, characterization, and absorption evaluation
Zafar et al. Formulation of intranasal surface engineered nanostructured lipid carriers of rotigotine: Full factorial design optimization, in vitro characterization, and pharmacokinetic evaluation
Mansour et al. Delineating the usage of dexamethasone-loaded cubosomes as a therapeutic armamentarium for hearing loss versus its protective effect: In-vitro and in-vivo animal study
Hoyos-Ceballos et al. Design of epigallocatechin gallate loaded PLGA/PF127 nanoparticles and their effect upon an oxidative stress model
Mittal et al. Quality by design engineered, enhanced anticancer activity of temozolomide and resveratrol coloaded NLC and brain targeting via lactoferrin conjugation in treatment of glioblastoma
Kaliamurthi et al. Insight on solid lipid nanoparticles: characterization and application in diabetes mellitus
Chinthaginjala et al. Nanostructured Lipid Carriers: A Potential Era of Drug Delivery Systems
Küçüktürkmen et al. A new approach for drug targeting to the central nervous system: Lipid nanoparticles
Chokshi et al. Fabrication and optimization of isoniazid loaded lipid nanoparticulate systems for the treatment of tuberculosis
Gazi et al. Preparation and evaluation of paracetamol solid lipid nanoparticle by hot homogenization method
Agrawal et al. Amelioration of bioavailability through formulating and optimizing Azilsartan Entrapped nanostructured lipid carriers and its pharmacokinetic assessment
WO2024095144A1 (en) Lipidic nanoparticles comprising an encapsulated drug and method of producing the same
Majeed et al. Formulation and in-vitro characterization of Capsaicin loaded ethosomes.
Eltawela Preparation and characterization of (−)-epigallocatechin gallate lipid based nanoparticles for enhancing availability and physical properties
Kunasekaran et al. Formulation and evaluation of nanoscale solid lipid particles containing a hydrophilic drug-rasagiline mesylate
Sahoo et al. A Focus on Fabrication, Characterization, Stability, Skin Targeting, Patent, Safety and Toxicity of Nanostructured Lipid Carrier
Kaushik Baicalein loaded polysorbate 80 nanostructured lipid carriers offered enhanced stability and in vitro drug release

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23813474

Country of ref document: EP

Kind code of ref document: A1