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WO2019068161A1 - Procédé d'obtention de transporteurs lipidiques nanostructurés, transporteurs lipidiques nanostructurés obtenus et utilisation de ceux-ci - Google Patents

Procédé d'obtention de transporteurs lipidiques nanostructurés, transporteurs lipidiques nanostructurés obtenus et utilisation de ceux-ci Download PDF

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WO2019068161A1
WO2019068161A1 PCT/BR2018/050364 BR2018050364W WO2019068161A1 WO 2019068161 A1 WO2019068161 A1 WO 2019068161A1 BR 2018050364 W BR2018050364 W BR 2018050364W WO 2019068161 A1 WO2019068161 A1 WO 2019068161A1
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oil
process according
lipid carriers
nanostructured lipid
rpm
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PCT/BR2018/050364
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English (en)
Portuguese (pt)
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Nádia ARACI BOU CHACRA
Paulo CESAR COTRIM
Lis MARIE MONTEIRO
Nikoletta FOTAKI
Raimar LÖBENBERG
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Universidade De São Paulo - Usp
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Application filed by Universidade De São Paulo - Usp filed Critical Universidade De São Paulo - Usp
Publication of WO2019068161A1 publication Critical patent/WO2019068161A1/fr

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    • 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
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is within the scope of medical science, more specifically, in the field of preparations for medical or veterinary purposes, since it relates to a process for the preparation of nanostructured lipid carriers comprising buparvaquone coated with cationic polymers and anionic compounds associated with a polypeptide, as well as nanocarriers obtained for the improvement of the treatment of leishmaniasis.
  • Leishmaniasis is among the major neglected tropical diseases. These diseases account for the second highest number of deaths due to parasitic infection in the world and are extremely associated with poverty. They are prevalent in 98 countries, on three of the five continents. About 1.3 million new cases occur annually and the estimated number of deaths from visceral leishmaniasis ranges from 20,000 to 50,000 per year.
  • the classical treatment of leishmaniasis requires the administration of poorly tolerated and highly toxic drugs, since the intracellular localization of leishmaniasis parasites hinders the access of chemotherapeutics to the site of action, which requires the administration of high and repeated doses of which is responsible for its high toxicity.
  • the pentavalent antimonials, meglumine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®) are the first-line compounds used to treat leishmaniasis.
  • the present invention proposes the development, optimization and evaluation of novel formulations of nanostructured lipid carriers coated with polymyxin B, chitosan and dextran for encapsulation of buparvaquone in order to improve the treatment of leishmaniasis.
  • nanostructured formulations comprising buparvaquone
  • IN02166MU2012A discloses a process for the preparation of solid nanoparticles (SLN) containing buparvaquone by ultrasound method.
  • the present invention proposes a method of preparing lipid carriers nanostructured (CLN) comprising buparvaquone by homogenization at high pressure; and the functionalization of these nanoparticles for site-specific release of the drug.
  • CLN are considered the second generation of lipid nanoparticles and are constituted by colloidal particles that present matrix composed by binary mixture of solid lipid with liquid lipid.
  • Such a special structure has advantages compared to SLNs, such as increased encapsulation efficiency, increased physical stability and reduced risk of drug release during storage.
  • the amount of drug can be increased by the use of liquid lipid, which buparvaquone has shown to be more soluble.
  • the present invention describes the coating of the nanoparticles, incorporating a second drug (polymyxin B) and molecules that enable drug internalization and release selectively into phagocytic cells. Additionally, it presents the CLN performance in parasites of Le ⁇ shman ⁇ a ⁇ nfantum and its safety using cytotoxicity tests.
  • US2003059470A1 describes the preparation of conventional, non-nanostructured emulsions comprising buparvaquone.
  • the CLN's consist of a mixture of solid lipid and liquid lipid. This blend provides novel properties, such as improved formulation stability and enhanced encapsulation efficiency.
  • US2003059470A1 teaches only conventional emulsions prepared with liquid lipid requiring only large amount of surfactants. Accordingly, the US document differs from the present invention in that it presents a formulation in the form of an emulsion which does not have any nanostructure, which has no site-specific action promoting the unexpected improved effect and the action of intracellular buparvaquone as proposed by the CLNs of the present invention.
  • the document BR102014023050A2 refers to the obtaining of lipid nanostructure employing high pressure homogenization and coating of this nanostructure employing polymyxin B.
  • polymyxin B cationic polymers
  • chitosan cationic polymers
  • extran anionic polymers
  • the present invention therefore provides the use of buparvaquone by means of nanocarriers in order to reduce the systemic toxicity of drugs present on the market, which would facilitate the acceptance of this drug by the patients to be treated and by the clinical staff.
  • the specific site action of the proposed buparvaquone CLNs is attributed to the coating by a combination of chitosan, dextran and dextran sulfate compounds, which allow the recognition by the cellular receptors of said CLN that allows site-specific action which promotes the unexpected improved effect and the action of intracellular buparvaquone.
  • the present invention relates to a method of obtaining nanostructured lipid carriers comprising buparvaquone coated with cationic and anionic polymers associated with a polypeptide by high pressure homogenization technology promoting the electrostatic interaction between its components in aqueous medium.
  • the present invention relates to such nanostructured lipid carriers obtained, which comprise from 0.5 to 50% w / v oil phase; 1 to 10% w / v surfactant; 50 to 500,000 IU / ml of a polypeptide; from 0.01 to 2% w / v of a cationic polymer; from 0.04 to 5% w / v of an anionic polymer; and purified water q.s.p.
  • the nanostructured lipid carriers obtained allow the site-specific release in macrophages, thus enabling the development of innovative drugs for the advancement of the treatment of leishmaniasis.
  • Figure 1 shows the diagram of steps ("g", “h”, “i") of the coating of nanostructured lipid carriers containing buparvaquone (A) by polymyxin B (B), chitosan (C) and dextran sulfate (D).
  • Figure 2 shows the flowchart of the process of obtaining the nanostructured lipid carriers of the present invention.
  • Figure 3 shows a photograph of the nanostructured lipid carrier formulation for the encapsulation of buparvaquone, wherein (A) refers to the formulation before and (B) refers to the formulation after high pressure homogenization.
  • FIG 4 graphically depicts the validation of the mathematical model of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone of formulation 1 (VI).
  • HVM mean particle hydrodynamic diameter
  • Figure 5 graphically depicts the validation of the mathematical model of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone of formulation 2 (V2).
  • HVM mean particle hydrodynamic diameter
  • Figure 6 shows the contour plots in the assay for evaluation of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone.
  • DLM mean particle hydrodynamic diameter
  • Figure 7 graphically represents the validation of the zeta potential mathematical model of nanostructured lipid carriers for the encapsulation of buparvaquone coated with polymyxin, chitosan and dextran of formulation 1 (VI).
  • Figure 8 graphically represents the validation of the mathematical model of the zeta potential of nanostructured lipid carriers for the encapsulation of buparvaquone coated with polymyxin, chitosan and dextran of formulation 2 (V2).
  • BPQ free buparvaquone
  • the present invention relates to a process for the preparation of nanostructured lipid carriers comprising buparvaquone coated with polymers cationic and anionic compounds associated with a polypeptide.
  • said method comprises the steps of:
  • step "a" the liquid and solid lipids are mixed in the preparation of the oil phase until homogenisation thereof.
  • said oily phase comprises 0.5 to 50% (w / v), preferably 5 to 15% (w / v), of a ratio of 0.1 (10: 1) to 10.0 (1 : 10) of liquid and solid lipids (LL / LS), respectively, preferably 0.5 (2: 1) to 3 (1: 3).
  • Liquid lipids are selected from a group consisting of caprylic and caprylic acid triglycerides, glyceryl monocaprylate, safflower oil, corn oil, olive oil, sesame oil, cottonseed oil, soybean oil, oleic acid, preferably triglycerides of capric and caprylic acids.
  • the solid lipids are selected from the group consisting of hydrogenated palm oil, hydrogenated coconut mono, di and triglycerides (Witepsol ® E85), stearoyl macrogol-32-glycerides (Gelucire ® 50/13), distearate of glyceryl (Precirol ® ATO 5), hydrogenated soybean oil (Sterotex ® HM), triglycerides of palmitic and stearic acids (Dynasan ® P60), glyceryl behenate (Compritol ® 888), preferably hydrogenated palm oil.
  • step "b" the buparvaquone is added in the oil phase obtained in the previous step, in the concentration of 0.01 to 5%, preferably 0.3% (w / v).
  • the aqueous phase which comprises the mixture of purified water qsp and from 1 to 10% (w / v) surfactant, is then prepared in step c, subjected to magnetic stirring until complete homogenization, preferably 2 to 4% (w / v).
  • the surfactants are selected from the group consisting of ethylene oxide and propylene oxide poloxamer copolymers 188, 407, 101, 105, 108, 122, 123, 124, 181, 182, 183, 184, 185, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403; sorbitan stearate, sorbitan monooleate, sorbitan trioleate, sorbitan tristearate, polysorbate 80, polysorbate 60, preferably poloxamer 188.
  • step "d" the mixture of the oily and aqueous phases is carried out at a temperature ranging from 45 to 85 ° C, preferably 70 ° C, with stirring ranging from 150 to 800 rpm, preferably 500 rpm, for 2 hours. to 45 minutes, preferably 10 minutes.
  • the preemulsion is formed by employing suitable equipment, such as a high performance disperser, at a rotation ranging from 500 to 14000 rpm, preferably 8000 rpm, at a temperature which varies 40-80 ° C, preferably 70 ° C, for 2 to 45 minutes, preferably 10 minutes.
  • suitable equipment such as a high performance disperser
  • step "f" the high pressure homogenization is performed for one to ten cycles IO 6 to IO 7 Pa, at the temperature ranging from 45 to 85 ° C, preferably 70 ° C.
  • step "g” the cooling is carried out at room temperature (20-25 ° C).
  • step "h” carrier dilution is performed in up to 1: 3 parts of purified water.
  • step "i" the addition of 50 to 500,000 IU / ml of a polypeptide, preferably 1000 to 9000 IU / ml, is added to the carrier at a temperature ranging from 20 to 25 ° C, homogenization by magnetic stirring ranging from 50 to 250 rpm, preferably 100 rpm, at a pH ranging from 3.5 to 9.0, preferably 5.0 to 7.0, for 50 to 120 minutes, preferably 60 minutes.
  • Said polypeptide is preferably polymyxin B and may be substituted for polymyxin E (colistin).
  • step "j" of 0.01 to 2% w / v of a cationic polymer, preferably 0.05 to 0.2% homogenization by magnetic stirring ranging from 50 to 1000 rpm, preferably 100 rpm, at a pH ranging from 3.5 at 9.0, at a temperature ranging from 20 to 25 ⁇ C, for 50 to 120 minutes, preferably 60 minutes.
  • Said cationic polymer is preferably chitosan, which may range from low to medium molecular weight (50 to 200 kDa), with a degree of deacetylation of at least 75%.
  • step "k" from 0.04 to 5% w / v, preferably from 0.4 to 0.55% w / v, of an anionic polymer is added to the homogenization by magnetic stirring which varies 50 to 150 rpm, preferably 100 rpm, at a pH ranging from 3.5 to 9.0, at a temperature ranging from 20 to 25 ⁇ C, for 50 to 120 minutes, preferably 60 minutes.
  • Said anionic polymer is preferably dextran sulfate, which may be substituted by D-mannose-6-phosphate.
  • step "1" the packaging of the obtained formulation is carried out for subsequent application in pharmaceutical vehicles.
  • Figure 2 shows the flowchart of the process of the present invention.
  • nanostructured lipid carriers comprising buparvaquone coated with cationic and anionic polymers associated with a polypeptide are obtained, enabling the recognition by their cellular receptors that they allow the site-specific action that promotes the effect and the action of intracellular buparvaquone in the treatment of visceral leishmaniasis and prevention of recidivism of cutaneous leishmaniasis.
  • the present invention relates to the obtained nanostructured lipid carriers which comprise:
  • Said oily phase comprises from 0.5 to
  • Liquid lipids are selected from the group consisting of triglycerides of caprylic and caprylic acids, glyceryl monocaprylate, safflower oil, corn oil, olive oil, sesame oil, cottonseed oil, soybean oil, oleic acid , preferably triglycerides of capric and caprylic acids.
  • Solid lipids are selected from the group consisting of hydrogenated palm oil, hydrogenated coconut mono, di and triglycerides (Witepsol ® E85), stearoyl macrogol-32-glycerides (Gelucire ® 50/13), distearate of glyceryl (Precirol ® ATO 5), hydrogenated soybean oil (Sterotex ® HM), triglycerides of palmitic and stearic acids (Dynasan ® P60), glyceryl behenate (Compritol ® 888), preferably hydrogenated palm oil.
  • said surfactant is in a concentration of 2 to 4% (w / v), and it is selected from the group consisting of ethylene oxide and propylene oxide (poloxamer) copolymers 188, 407, 101, 001, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403; sorbitan stearate, sorbitan monooleate, sorbitan trioleate, sorbitan tristearate, polysorbate 80, polysorbate 60, preferably poloxamer 188.
  • said polypeptide is in a concentration of 1000 to 9000 IU / ml, which is preferably polymyxin B and may be substituted for polymyxin E (colistin).
  • said polymer cationic acid is in a concentration between 0.05 and 0.2%, which is preferably chitosan, which may range from low to medium molecular weight (50 to 200 kDa), with a degree of deacetylation of at least 75%.
  • the anionic polymer is in a concentration of 0.4 to 0.55% w / v, which is preferably dextran sulfate, which may be substituted by D-mannose-6-phosphate.
  • said nanostructured lipid carriers have a mean hydrodynamic diameter (DHM) ranging from 100 to 350 nm; zeta potential (mV) less than -10, preferably less than -20; and polydispersity index less than 0.3.
  • HDM mean hydrodynamic diameter
  • mV zeta potential
  • said nanostructured lipid carriers have novel features that allow nanoparticles to be internalized in the macrophage cell membrane through the direct action of polymyxin B, combined with the interaction of polysaccharides with SIGN-R1 receptors.
  • administration of such carriers directed to the lymphatic system may be an innovative approach for the treatment of leishmaniasis.
  • the present invention relates to the use of the nanostructured lipid carriers obtained for the manufacture of a medicament for treating leishmaniasis.
  • Example of the invention [064] Hydrogenated palm oil (Softisan ® 154) was chosen for the preparation of the nanoparticles, since buparvaquone (BPQ) presented greater solubility in this lipid and because its melting point varies from 53-58 ° C, characteristic that allows the addition of a greater amount of liquid lipid to the solid lipid while maintaining the solid characteristics of the mixture.
  • BPQ buparvaquone
  • caprylic and caprylic acid triglycerides have been shown to be lipid which best solubilizes BPQ, which lipid is widely used for oral and topical formulations, is resistant to oxidation and is synthesized industrially and, therefore, was chosen as liquid lipid to compose formulations of CLNs.
  • the first step in the preparation of the CLNs consisted of heating and stirring the oily and aqueous phases at 70 ⁇ 5 ° C, at 500 ⁇ 50 rpm for 10 minutes.
  • the second step consisted of pre-emulsion formation using ultra-turrax disperser at 8,000 rpm for 10 minutes.
  • THE third and final stage consists of homogenization at high pressure for five cycles at 600 bar and 70 ⁇ 5 ° C.
  • LS solid lipid.
  • LL liquid lipid.
  • LS / LL ratio of solid and liquid lipid
  • GL degrees of freedom
  • CT contribution
  • SQ seq sum of squares
  • SQ aj sum of squares adjusted
  • Test F statistics F
  • MQ (aj) adjusted quadratic mean.
  • P-value level of significance.
  • Equation 1 that describes the influence of the factors in the process is presented below. With this equation it is possible to calculate the DHM values from the variation of the concentrations of poloxamer, oil phase and LS / LL.
  • POL poloxamer concentration (w / v);
  • FO oily phase concentration (w / v).
  • Formulations VI and V2 having the parameters shown in Figures 6 and 7. These formulations present optimized excipient concentrations to obtain DHMOOO nm.
  • FIG. 4 shows the graph of formulation validation 1 (VI) of the mathematical model of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone.
  • V2 the modified parameter was the 2% (w / w) poloxamer concentration.
  • the model provided the theoretical DHM value of 213.7 nm with a confidence interval of 189.4 to 238.0 nm (Table 3).
  • Figure 5 shows the graph of the validation formulation 2 (V2) of the mathematical model of the mean particle hydrodynamic diameter (DHM) of nanostructured lipid carriers for the encapsulation of buparvaquone.
  • Table 3 Validation of the mathematical model of the mean particle hydrodynamic diameter of the production process of nanostructured lipid carriers for the encapsulation of buparvaquone.
  • the concentrations of the materials should be among the studied tracks.
  • the range should be 0.5 (2: 1) to 3 (1: 3).
  • the range should be between 5 to 15% (w / v).
  • the poloxamer concentration it should remain between 1 and 4% (w / v), as previously described.
  • the first step of the coating is the addition of polymyxin B solution to the carrier in order to deposit the drug on the surface but to avoid the complete neutralization of negative charges from the carrier.
  • Chitosan PM: 50000-150000 g / mol, 75-90% deacetylation
  • presents positive charges which interact with the liquid negative charges of the first step, by reversing the zeta potential to positive.
  • the third step is similar to that described in the literature, it relates to the preparation of particles formed with chitosan nucleus and coated with dextran. Due to the positive net charge of the carriers, dextran sulfate (PM ⁇ 40,000 g / mol), of negative charge, can be deposited on the surface of the nanostructured lipid carrier.
  • dextran sulfate PM ⁇ 40,000 g / mol
  • the coating process was started by preparing stock solutions of polymyxin B: 100,000 IU / ml, chitosan: 2% (w / v) and 2% dextran sulfate (w / v) and the dilution of a part of the nanostructured carrier to three parts purified water.
  • the first step consists of the addition of the solution of polymyxin B to the diluted carrier, the homogenization was carried out by magnetic stirring at 100 rpm for 1 hour.
  • the chitosan is added and the stirring is continued for another hour.
  • the dextran sulfate is added and the system is maintained for another hour at 100 rpm.
  • a small aliquot is removed for verification of the zeta potential as process control.
  • the coating process was carried out in the following conditions: pH 5, 0 - 7.0, temperature 25 ° C.
  • Table 5 Analysis of variance to test the significance of the regression for the data obtained in the assay for the evaluation of the zeta potential of nanostructured lipid carriers for the encapsulation of buparvaquone coated with polymyxin, chitosan and dextran.
  • GL degrees of freedom
  • SQ seq sum of squares
  • SQ aj sum of squares adjusted
  • Test F statistics F
  • MQ (aj) adjusted quadratic mean.
  • P-value level of significance.
  • Table 6 addresses the interaction between chitosan and dextran. This interaction contributes to the reduction of zeta potential (negative coefficient -4,24). This result reveals how the application of a factorial study is a necessary tool to describe and understand a process or product and even to provide a theoretical basis for the continuous improvement of these.
  • Equation 2 presents the mathematical model that describes the coating process of the nanostructured lipid carrier for encapsulation of buparvaquone. This equation was used to optimize this process in order to reduce the zeta potential to approximately - 30 mV. These formulations with reduced zeta potential have loads necessary for repulsion between particles, which contributes to the long-term stability of the coated carriers.
  • PZ zeta potential in mV (millivolts).
  • POL concentration of polymyxin B in IU / mL
  • QUI chitosan concentration in% (w / v); and DEX: dextran concentration in% (w / v).
  • Figure 6 shows the behavior of the zeta potential for each pair of factors.
  • the graphs show that the concentration of chitosan should be less than 0.2% w / v and that of dextran should be above 0.4% w / v for formulations of zeta potential less than -20 mV can be achieved. Therefore, in the preparation of coated CLNs, the concentrations should be the following, as previously described: polymyxin B between 1,000 and 9,000 IU / ml, chitosan between 0.05 and 0.2% (w / v) and dextran between 0.4 to 0.55% (w / v).
  • Table 7 Validation of the zeta potential (PZ) model of the production process of nanostructured lipid carriers for the encapsulation of buparvaquone coated with polymyxin, chitosan and dextran.
  • Figures 7 and 8 present, respectively, formulations VI and V2 for the validation of the mathematical model in the coating process of nanostructured lipid carriers with polymyxin B, chitosan and dextran.
  • Said concentrations refer to the optimized formulations.
  • the reduced concentration of chitosan in V2 reflects calculated zeta potential lower than VI.
  • Dextran concentrations were maintained at the upper limit of the model to obtain carriers with the lowest possible zeta potential.
  • Table 7 shows the results practical and theoretical aspects of validation formulations. In both formulations, the value obtained was within the predicted range (95% confidence interval). Therefore, the mathematical model is validated and can be used to predict zeta potential values according to the modifications in the desired main factors.
  • Encapsulation efficiency was calculated by subtracting the amount of drug in the supernatant from the total drug concentration in the sample. Two uncoated samples were tested and the results are summarized in Table 8 and show that the encapsulation efficiency was satisfactory because it has values close to 100%.
  • the evaluation of cytotoxicity was performed in two cell types, THP-1 and mouse peritoneum macrophages.
  • the first cell type was cultured in RPMI 1640 medium and treated with PMA (phorbol myristate acetate) ⁇ for 24 hours for monocyte differentiation to human macrophages.
  • PMA phorbol myristate acetate
  • 2x 10 5 cells were incubated for 24 h at 37 ° C with final formulation VI.
  • the concentrations used were 0.88; 1.75; 3.5; 7.0; and 14.00 ⁇ b buparvaquone.
  • Table 11A shows the solubility of uncoated CLNs.
  • the coated nanoparticles should be employed in injectable formulations. All formulations showed higher solubility in water when compared to free drug. In general, the lower the DHM, the greater the solubility.
  • Formulation VI which has the lowest DHM (170.4 nm), showed solubility of 611 times greater when compared to the free drug in the simulated body fluid. Even in the presence of high concentration of sodium dodecyl sulfate (1% w / v) pH 7.4, the solubility was 3.2 times higher.
  • Table 12 shows the dissolution values of free BPQ in pH 7.0, pH 4.0, 0.05M phosphate buffer with 0.07% w / v Tween 80. Even after four hours, only 2.89% of 4 mg of free drug was dissolved. Such a result revealed that the drug did not reach the solubility value (3.39 g / ml) in that medium. During the development of the dissolution method, pancrelipase was tested to mimic the degradation of the nanoparticles from intestinal lipases.
  • Figure 9 shows the dissolution profiles in 0.05M phosphate buffer pH 7.4 with 0.07% tween 80 of formulations VI and V2. Table 13 and Table 14 show mean dissolution values.
  • Free BPQ in pH 4.0, 4.00M phosphate buffer with 1% (w / v) sodium dodecyl sulfate Although the concentration of the surfactant increased when compared to the previous method (tween 80 0.07%), the free drug did not dissolve in this medium. This can be explained by the precipitation of BPQ due to interaction with the sodium salt, which may occur in vivo. Therefore bile salts, such as sodium taurocholate, can precipitate the drug in the gastrointestinal system.
  • Figure 10 and Table 16 show the dissolution profiles of formulations VI and V2 in pH 7.0, 4.00 M phosphate buffer with 1% (w / v) sodium dodecyl sulfate.
  • VI the dissolution reached 83.71% at 5 minutes of the test, although some precipitation was observed at 20 minutes, the dissolution was 75.12%.
  • the extent of precipitation and the variation between the samples (DP) were minimized when compared to the Tween 80 medium profiles.
  • the dissolution reached 79, 84% of the 4 mg dose after 60 minutes. As found in VI, precipitation and standard deviation were reduced.
  • the formulations developed may constitute a drug delivery system directed to the lymphatic system.
  • the drug can reach the lymphatic vessels due to the small size of the nanoparticles or even the drug dissolved in the lipids, which can be hydrolyzed by pancrelipase.
  • Drug administration directed to the lymphatic system may be an innovative approach for the treatment of leishmaniasis. After the bite, the parasites are disseminated through the vascular and lymphatic systems, and infect monocytes and macrophages of the mononuclear system. The spleen, liver and lymph nodes are the organs most affected by visceral leishmaniasis. Thus, absorption and distribution of BPQ through the lymphatic system may increase drug availability at the site of action.
  • Table 17 shows the EC 50 values for each condition. All presented improved leishmanicidal activity when compared to free BPQ. VI showed EC 50 of 229.0 nM, which represents a 2.0-fold increase. The coated formulation showed increase in EC 50 (150.5 nM) 3.0 fold compared to free BPQ (456.5 nM). This difference can be explained by the direct action of polymyxin B, combined with the interaction of polysaccharides with SIGN-R1 receptors on the macrophage cell membrane, which would favor internalization of the nanoparticles.
  • Endocytosis is a process of eukaryotic cells, which consists of the internalization of extracellular substances typically by the invagination of the membrane forming vesicles, known as phagosomes. After internalization, the lysosome fuses with the phagosome and releases its hydrolases to degrade the content in amino acids, fatty acids and glucose. This resulting structure is known as phagolysosome.
  • CLNs Due to the results of the present invention, developed CLNs have potential application to improve drug efficacy, reduce treatment toxicity, and improve patient compliance. Such formulations may be used as oral and parenteral medicaments to fill the gaps of conventional treatment of leishmaniasis. As a consequence, the cost can be minimized by reducing hospitalization for medication administration and monitoring of side effects.

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Abstract

La présente invention concerne un procédé d'obtention de transporteurs lipidiques nanostructurés comprenant de la buparvaquone revêtus de polymères cationiques et anioniques associés à un polypeptide au moyen de la technologie d'homogénéisation à haute pression, favorisant l'interaction électrostatique entre ses composants en milieu aqueux. En outre, la présente invention concerne lesdits transporteurs lipidiques nanostructurés obtenus, lesquels renferment entre 0,5 et 50% p/v d'une phase huileuse, entre 1 et 10% p/v d'un tensioactif, entre 50 et 500000 Ul/ml d'un polypeptide, entre 0,01 et 2% p/v d'un polymère cationique, entre 0,04 et 5% p/v d'un polymère anionique, et de l'eau purifiée en quantité suffisante pour permettre, ainsi, une libération sitio-spécifique dans des macrophages, permettant l'élaboration de médicaments innovants constituant une avancée dans le traitement de la leishmaniose.
PCT/BR2018/050364 2017-10-04 2018-10-03 Procédé d'obtention de transporteurs lipidiques nanostructurés, transporteurs lipidiques nanostructurés obtenus et utilisation de ceux-ci WO2019068161A1 (fr)

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Citations (3)

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BR102014023050A2 (pt) * 2014-09-17 2016-04-12 Univ Sao Paulo processo de obtenção de um sistema nanoestruturado catiônico, sistema nanoestruturado catiônico e seu uso
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BR102014007923A2 (pt) * 2014-04-02 2016-04-12 Univ Sao Paulo sistema nanoestruturado polimérico e seu uso
BR102014023050A2 (pt) * 2014-09-17 2016-04-12 Univ Sao Paulo processo de obtenção de um sistema nanoestruturado catiônico, sistema nanoestruturado catiônico e seu uso

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