WO2015177820A1 - Systèmes nanoparticulaires visant à véhiculer des médicaments pour le traitement de pathologies liées à une infection par leishmania - Google Patents

Systèmes nanoparticulaires visant à véhiculer des médicaments pour le traitement de pathologies liées à une infection par leishmania Download PDF

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WO2015177820A1
WO2015177820A1 PCT/IT2015/000134 IT2015000134W WO2015177820A1 WO 2015177820 A1 WO2015177820 A1 WO 2015177820A1 IT 2015000134 W IT2015000134 W IT 2015000134W WO 2015177820 A1 WO2015177820 A1 WO 2015177820A1
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nanoparticles
polylactic acid
nanocapsules
cells
antimonial
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PCT/IT2015/000134
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Domenico BRITTI
Donato Cosco
Massimo Fresta
Donatella Paolino
Elena TRAPASSO
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Università Degli Studi "Magna Graecia" Di Catanzaro
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • 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
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • 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 relates to nanoparticle systems as carriers of drugs for the treatment of pathologies related to the infections with Leishmania.
  • the invention relates to nanoparticle systems of polymeric nature as effective vehicles of compounds based on antimony, also antimonials, for example N-metilglucamine antimoniate (Glucantime ® ). More in particular, the more used active principle for the treatment of pathologies related to infection with Leishmania, was encapsulated into the aqueous "core" (internal part) of nanocapsules enclosed within an external layer or polylactic acid (PLA) "shell".
  • PPA polylactic acid
  • Said nanocapsules allow reducing the toxicity of the antimonial compounds in the cells and modulating their biopharmaceutical properties while keeping their pharmacological effectiveness unchanged.
  • Leishmaniasis is a disease caused by protozoan parasites belonging to the genus Leishmania and that are spread by biting insects belonging to the subfamily Phlebotominae, among which the insects known in Italy with the name "pappataci".
  • the protozoan spread by the insect bite propagate inside the reticuloendothelial cells of the guest (macrophages, reticular spleen cells, lungs, bone marrow and lymphnodes, Kupfer cells in the liver) in its replicating intracellular form (amastigote). It is calculated that about 12 million people in the world suffer from leishmaniasis.
  • Leishmaniasis can affect both animals and humans and can be divided into three different types:
  • Cutaneous leishmaniasis the form that most commonly develops at the bite site. It takes a long time to heal and leaves very visible scars. This form can progress into:
  • Antimonials are pharmaceutical compounds based on pentavalent antimony (Sb v ) that are the standard treatment for the cure of canine and human leishmaniasis even though they are extremely toxic compounds.
  • Sb v pentavalent antimony
  • MA meglumine antimoniate
  • Pentostam ® a pentavalent antimony compound having properties similar to those of another pentavalent antimony compound, i.e., sodium stibogluconate
  • Kala-azar black fever
  • Leishmania donovani complex L. donovani donovani, L. infantum e L. chagasi.
  • this compound is also used in the treatment of cutaneous leishmaniasis caused by different species among which L. major, L. tropica e L. braziliensis.
  • the antimonials' mechanism of action is linked to the inhibition of the parasite's glycolytic activity and fatty acid oxidation inhibition, thus favoring a reduction in the antioxidant defensive mechanisms and the depletion of the ATP reserves that represent the energy needed for the parasite's survival (Frezard et al., 2009).
  • the side effects that appear after the therapy with meglumine antimoniate are tubular renal insufficiency (as a consequence of the decreased drug clearance, in this regard see Ikeda-Garcia et al., 2007; Bianciardi et al., 2009; de Moura et al., 2008), liver and pancreatic function alterations and cardiac arrhythmias (Berman, 1997).
  • the polymeric nanoparticles offer the possibility of making nanosystems that are able to modify the biopharmaceutical features of the enclosed compounds (Pitarresi et al., 2007; Trapani et al., 2011), reducing their toxic side effects, as the result of a decrease in the number of administrations and of a better pharmacological action (Laquintana et al., 2009; Acharya e Sahoo. 2011; Celia et al., 2011).
  • the molecular weight of the polymer, for example PLA or PLGA, used to make the nanocapsules can deeply influence important chemical, physical, and biological properties of the particles obtained, such as, for example, the Entrapment Efficiency and the ability to carry their contents into cells (Marchais et al., 1998; Zambaux et al., 1999; Li et al., 2013).
  • the preparation method of the nanoparticles and the chemical nature of the compounds to be encapsulated influence their useful features for the pharmacological use.
  • polylactic acid-based nanocapsules are known as the vehicle for compounds that are very different from antimonials, for example gemcitabine hydrochloride (Paolino et al., 2012) and insulin (Yinyan Zhu et al, 2005).
  • polylactic acid PLA has been approved by Food and Drug Administration (Anderson and Shive. 1997).
  • MA was encapsulated in a liposomal formulation of distearoylfosphatidylcoline, cholesterol and diacetyl phosphate (molar ratio 5:4:1), obtaining a reduction in the toxicity in mongrel dogs suffering from visceral leishmaniasis (Ribeiro et al., 2008).
  • Navaei et. al. (2014) and WO 2013/010238A1 describe the preparation of PLGA microspheres that carry antimonial compounds. Such microspheres, anyway, due to their dimensions, are not useful for the parenteral use, in particolar the endovenous one, as is instead possible using nanoparticles such as those of the invention.
  • the object of the present invention a polymeric nanoparticle system of polylactic acid that efficiently encapsulates antimonial drugs, in particular N-methylglucamine antimoniate (from now on also MA or Glucantime ® ) in systems made of an aqueous nucleus (core) in which the active principle is entrapped, surrounded by a polymeric biocompatible and biodegradable shell based on polylactic acid, so that the compound is carried and concentrated within the guest's macrophages, thus avoiding the appearance of the typical toxic systemic effects linked to the therapy with antimonial drugs.
  • antimonial drugs in particular N-methylglucamine antimoniate (from now on also MA or Glucantime ® )
  • core aqueous nucleus
  • the active principle is entrapped
  • a further object are the pharmaceutical compositions comprising the nanoparticle system carrying drugs for the treatment of leishmaniasis, in particular antimonials, more in particular MA, and suitable excipients pharmaceutically approved.
  • biocompatible and biodegradable polymer nanoparticles based on polylactic acid of the invention for the treatment and prevention of the spreading of pathologies related to Leishmania infections in animals and humans.
  • FIG. 1 Entrapment Efficiency (EE) of pentavalent antimony (Sb v ) into polylactic acid nanocapsules containing sodium deoxycholate or Tween 80 (TN80).
  • EE Entrapment Efficiency
  • FIG. 1 Transmittance and backscattering profiles for formulation A (empty polylactic acid noanocapsules) obtained with Turbiscan Lab ® Expert. Data are shown as a time function (0-1 h) and depth of the sample (from 2 to 10 mm).
  • Figure 3. Transmittance and backscattering profiles for formulation B (polylactic acid noanocapsules prepared with 1 mg MA) obtained with Turbiscan Lab ® Expert. Data are shown as a function of time (0-1 h) and depth of the sample (from 2 to 10 mm).
  • FIG. 4 Transmittance and backscattering profiles for formulation F (polylactic acid noanocapsules prepared with 5 mg MA) obtained with Turbiscan Lab ® Expert. Data are shown as a function of time (0-1 h) and depth of the sample (from 2 to 10 mm).
  • FIG. 5 Transmittance and backscattering profiles for formulation G(polylactic acid noanocapsules prepared with 10 mg MA) obtained with Turbiscan Lab ® Expert. Data are shown as a function of time (0-1 h) and depth of the sample (from 2 to 10 mm).
  • Figure 6 Release profile of Sb from formulation G as a function of time. The release experiment were performed in sink conditions at the thermostatted temperature o 37°C.
  • Figure 7 In vitro cytotoxicity of polylactic acid nanoparticles in J774A.1 cells, as a function of polymer concentration and of exposure time (Figure 7A: 24h, Figure 7B 48h, Figure 7C 72h). Data are expressed as the percentage of cell viability measured by means of the MTT assay. The results are the average of four different experiments ⁇ standard deviation. The error bars, if not shown, are within the symbols.
  • Figure 8 Interaction levels between polylactic acid nanoparticles radiolabeled with [ 3 H]CHE and J774A.1 cells.
  • Figure 9 Intracellular accumulation of Sb administered as MA (Glucantime) or as polylactic acid nanocapsules loaded with MA into J774A.1 cells as a function of the incubation time. The experiments were performed at 37°C at a final concentration of 10 ⁇ of the drug. Each bar is the average of five different experiments ⁇ standard deviation.
  • Figure 10 CLSM microphotographs of untreated J774A.1 cells.
  • Panel A FITC filter
  • panel B Hoechst filter
  • FIG. 11 CLSM microphotographs of J774A.1 cells (left) treated with polylactic acid nanocapsules labelled with DMPE- rhodamine after a 4-h incubation (right).
  • Panel A TFJTC filter;
  • panel B Hoechst filter;
  • Figure 13 CLSM microphotographs of J774A.1 cells treated with nanocapsules of polylactic acid labelled with rhodamine-DMPE after a 4-h incubation, maximum enlargement to appreciate the intracellular localization of the systems.
  • Panel A TRITC filter
  • panel B Hoechst filter.
  • Figure 14 Microscopy images of J774A.1 cells treated with nanocapsules of polylactic acid labelled with rhodamine-DMPE after a 1-h incubation, section at different depths.
  • Panel A TRITC filter
  • panel B Hoechst filter
  • panel C superimposition.
  • Figure 15. CLSM microphotographs of J774A.1 cells (50X) infected by parasites.
  • Panel A untreated cells.
  • Panel B cells treated with Glucantime (free MA) at 50 ⁇ Sb concentration.
  • Panel C cells treated with Glucantime (free MA) at 10 ⁇ Sb concentration.
  • Panel D cells treated with nanocapsules loaded with MA at 50 ⁇ Sb concentration.
  • Figure 16 Microscopy images of J774A.1 cells infected with parasites after Giemsa dyeing (50X).
  • Panel A untreated cells.
  • Panel B cells treated with Glucantime (free MA) at 50 ⁇ Sb concentration.
  • Panel C cells treated with polylactic acid nanocapsules charged with MA at 50 ⁇ Sb concentration.
  • Figure 17 Biodistribution of polylactic acid microcapsules radiolabeled with [ 3 H]CHE in B57 mice as a function of time.
  • FIG. 18 Biodistribution of Sb v (2.3 mg/kg) in B57 mice after i.v. administration of the antimonial compound MA (Glucantime).
  • Figure 19 Biodistribution of Sb v (2.3 mg/kg) in B57 mice after i.v. administration of polylactic acid nanocapsules charged with MA.
  • the present invention relates to the encapsulation of antimony- based compounds.
  • antimonials such as, for example, Glucantime ® and Pentostam ®
  • polylactic acid nanoparticles to obtain a more efficient way to carry this active principle within the cells and the organs infected with parasites of the genus Leishmania in humans and animals, in particular dogs, and, at the same time, to decrease its systemic toxicity.
  • Particularly preferred is MA.
  • nanocapsules and “nanoparticles” are used as synomims and refer to particles having in general dimensions from 900 to 20 nm, preferably 800-50 nm.
  • the nanoparticles according to the invention are made of an external polylactic acid (PLA) layer or “shell” and an inner part or “core” that comprises an antimonial compound in aqueous solution.
  • PVA polylactic acid
  • Polylactic acid has an average molecular weight of 20-200 kDalton, preferably 20-160 kDalton,, more preferably 85-160 kDalton.
  • PLA low molecular weight PLA
  • the encapsulation process of the invention comprises the following steps:
  • the preparation of the aqueous suspension can be carried out, for example, by means of sonication in ice bath or homogenization.
  • the non-ionic lipophilic detergent can be chosen from Span 80, 60, 40 and can be added up to a concentration of, 0.1-5% w/v;
  • the organic solvent can be acetone, hexane, tetrahydrofurane (THF), ethyl ether, methanol, dichloromethane, chloroform and mixtures thereof with the aim to obtain a homogeneous dispersion of the organic phase in a polar milieu, in particular an aqueous milieu.
  • the homogenization can be carried out, for example, with Ultraturrax.
  • the non-ionic hydrophilic detergent can be Tween 80, 60, 40 e 20 sodium deoxycholate, polyols such as glycerol, propylene glycol, sorbitol, mannitol and mixtures thereof.
  • solvent evaporation can be carried out at room temperature.
  • the average size of the particles increased with the increasing of the amount of MA present
  • the decrease in the variability of ⁇ potential points to a noteworthy stability of the particles themselves.
  • the ⁇ potential is a parameter used to identify the superficial charge in systems of particles.
  • the entrapment efficiency (EE) of increasing amounts of the active principle MA by the nanoparticles prepared in the presence of 1% SD w/v, used in the second aqueous suspension step is higher with respect to the nanoparticles formulated using Tween 80 at the same concentration.
  • the nanoparticles' stability was studied using the Turbiscan Lab ® Expert, a tool that allows relating the stability of a colloidal system, in this case the nanoparticle system of the invention in aqueous solution, to its backscattering (BS) and transmittance (T) features (Celia et al., 2009).
  • BS backscattering
  • T transmittance
  • Figure 2 are shown the profiles for formulation A (empty nanospheres of the invention); in Figure 3 are shown the profiles for formulation B (nanospheres of the invention prepared with 1 mg of MA; in Figure 4 are shown the profiles for formulation F prepared in the presence of 5 mg MA and in Figure 5 are shown the profiles for formulation G prepared in the presence of 10 mg MA, respectively.
  • the results obtained show that the nanocapsules 1 structure allows entrapment of great amounts of MA since the BS and T profiles do not show significant variations ( ⁇ 5%) with respect to those obtained by testing the empty formulation.
  • Figures 2-5 thus clearly show that the polymeric nanocapsules of the invention correspondent to formulations B, F and G when compared with formulation A (nanoparticles not containing the active principle) can efficiently encapsulate MA without showing destabilization phenomena, as demonstrated by 5% both negatively and positively in all the analyzed samples).
  • the profile for the antimony release by the nanoparticles of formulation G of the invention is shown in Figure 6.
  • the release was measured by means of the dialysis method, as described herein below in the "Examples" section, for 24 hours. As shown in said Figure, it is possible to notice a peculiar first stage of fast release after 4 hours (about 50%), followed by a slower and more gradual stage.
  • Figure 8 show that the nanoparticles of the invention loaded with an amount equivalent to 10 ⁇ of pentavalent antimony, reach the best interaction level with the cells after 2 hours and keep it for more than 12 hours. After 24 hours of incubation this interaction wanes, probably due to the nanocapsules metabolization by the cells themselves.
  • FIG 14 a CLSM microphotograph of J774A.1 cells treated for 1 hour with the nanoparticles of the invention that encapsulated the dyes rhodamine and Hoechst. It is evident that the efficiency of the interaction between the particles and the cells is such that, even after a short incubation period, the cell cytoplasm already presents with a diffuse fluorescence that does not involve the nucleus.
  • Vehiculation by means of the nanocapsules of the invention brings about a remarkable decrease of the pentavalent antimonial compound vs. an increase in effectiveness, as demonstrated by the following results.
  • Toxicity toward macrophage cells was studied in J774A.1 infected with L. tropica and dyed with the Giemsa technique.
  • the empty nanocapsules labelled with [ 3 H]CHE-polylactic acid administered into the caudal vein rapidly and preferentially are found in the reticulo-endothelial system (RES) cells of liver, spleen and lungs, rapidly disappearing from the bloodstream.
  • RES reticulo-endothelial system
  • the Encapsulation Efficiency is at least 50%, preferably 60%, more preferably 70% with respect to the antimonial present in the preparation mixture of the nanoparticles of the invention.
  • the encapsulation efficiency obtained using PLA of molecular weight ⁇ 20kDalton was, on the contrary, ⁇ 10% with respect to the antimonial used for the preparation of the nanocapsules.
  • the nanoparticles of the invention containing MA effectively interact with macrophages, without damaging them or not altering in a significant way their morphology and functioning.
  • the nanoparticles of the invention allow preserving the pharmacological activity of the active principle MA by decreasing its accumulation in heart and kidney tissues, which cause serious side and undesired affects seen in vivo, even though they cause, with respect to the non-encapsulated antimonial derivative, a prolonged presence in the bloodstream with respect to the antimonial derivative chosen.
  • the antimonial active principle vehiculated with polymeric biodegradable polylactic acid nanoparticles according to the present invention can be conveniently formulated as known to the expert in the field for the paranteral administration.
  • the amounts of antimonial active principle that can be encapsulated and that can be effective for the treatment of pathologies related to the infection with Leishmania as described above are the same or are lower than the ones currently used for marketed antimonial products that range from 15 to 100 mg kg.
  • the encapsulated antimonial concentration range is 1-7 mg.
  • the antimonial compound in the aqueous core of the nanoparticles of the invention, can be advantageously associated with other active principles that potentiate its pharmacological effects, thus obtaining a co-delivery system.
  • active principles can, alternatively, be administered in association with the nanocapsules of the invention for a combined, simultaneous, sequential or retarded administration.
  • An example of such different active principles is amphotericin B.
  • parenteral refers to the administration of medicaments by injection through the teguments or directly in the bloodstream.
  • parenteral administration are the administration by intradermal route (i.d.), subcutaneous route (s.c), intramuscular route (i.m.) and endovenous route (e.v.).
  • the formulations containing the nanocapsules of the invention can comprise carriers (for example, water, saline solution, byffer solutions, oil components); stabilizers (for example, antioxidants);
  • chelating agents for example EDTA
  • antimicrobial agents for example EDTA
  • the nanocapsules of the invention can be used for the treatment of pathologies related to the infection with Leishmania in animals and humans, such as those caused by Leishmania donovani complex: L. donovani donovani, L. infantum, L. chagasi, L. major, L. tropica e L. braziliensis.
  • the pathologies in particular are: cutaneous leishmaniasis, diffuse cutaneous leishmaniasis, mucocutaneous leishmaniasis, visceral leishmaniasis.
  • nanocapsules also allows prevention or reduction of the side effects of traditional antimonials, linked in particular to the accumulation of SB V at the level of kidney and heart tissues.
  • the nanocapsules can advantageously be administered in suitable dosages that the physician will choose in relation with the subject to be treated, of his physical conditions and of the seriousness of the pathology.
  • Subjects that can be treated are humans and animals. Animals can be chosen from pets and wild animals, such as: dogs, cats, horses, ferrets, weasels, foxes, pigs, and from carnivorous birds, such as birds of prey. The following examples are to be considered as illustrative and not as limiting the scope of the invention.
  • Poly-L-lactide (MW 85,000-160,000), hydrated sodium deoxycholate (SD), dimirystoyl-phospatidyl- ethanolamine, sulforhodamine B (DMPE-rhodamine), glycerol, phosphate buffered saline solution tabs, 3-[4,5- dimethylthiazole-2-yl]-3,5-diphenyltetrazole bromide salt (used for the MTT assays), dimethylsulphoxide and amphotericin B solution (250 pg/ml) were purchased from Sigma Aldrich (Milan, Italy).
  • Sorbitan esters (Span ® ) and polysorbates (Tween ® ) were purchased from ACEF S.p.A. (Piacenza, Italy).
  • Cells J774A.1 were purchased from Banca Biologica and Cell Factory - 1ST National Institute for Cancer Research (Genoa, Italy).
  • Exadecyl [ 3 H]-colesteryl ether ([ 3 H]- CHE, 40 Ci/mmol) was purchased from Perkin Elmer (Monza, Italy).
  • D-MEM Dulbecco's modified essential medium
  • FBS fetal bovine serum
  • Ix trypsin-EDTA solution
  • penicillin- streptomycin solution 100 UI/mL
  • the nanoparticles prepared as described above, were used suspended in distilled water or, for the experiments with cells and for the in vivo experiments, in saline solution (0,9% w/v).
  • the nanoparticles in aqueous solvent form colloidal systems, i.e. suspensions characterized by an average diameter of the solid phase components equal to or lower than 500 nm, preferably 100-350 nm.
  • Example 1 Preparation of Polymeric Nanoparticles
  • the polymeric nanocapsules were obtained by means of the method previously described in Paolino et al. (2012). Briefly, 1 ml of aqueous solution (containing different amounts of Glucantime, MA) was added to a polylactic acid (6 mg), Span (1% w/v), chlorophorm (2.5 ml) mixture and was sonicated for 2 minutes using a Sonopuls GM70 (Bandelin) at 30% of the maximal potency in an ice bath.
  • aqueous solution containing different amounts of Glucantime, MA
  • the suspension obtained was added to an aqueous solution containing Tween 80 or SD (1% w/v) andf glycerol (1 ml) and homogenized at 24000 rpm for 1 minute with an Ultraturrax T25, IKA ® Werke (Germany).
  • the formulation was kept for 3 hours under stirring on a magnetic stirrer to allow solvents evaporation and the formation of the particellar systems.
  • Nanoparticles containing Glucantime were prepared by adding different amounts of the drug (1, 2, 3, 4, 5 and 10 mg of MA) to the first aqueous phase. Nanoparticles containing Pentostam ® were prepared the same way.
  • the fluorescent nanoparticles were prepared dissolving the rhodamine- DMPE (0,1% molarity) with the polymer in the organic phase during the preparation of the sample.
  • the aqueous solution used for the preparation of the nanoparticles was a saline solution (NaCl 0,9% w/v) to the end of avoiding osmotic stresses to the particles structure.
  • the average distribution of the size and of the Z potential were tested with a Zetasizer Nano ZS (Malvern Instruments Ltd., Worchester shire, Great Britain) apparatus based on the technology of light diffusion applied to microelectrophoresis, applying the third order cumulative adaptation function.
  • a laser 4,5 mW diode operating at 670 nm was used as the light source for the size analysis and the reflected photons were detected at 173°.
  • the actual and imaginary index were set at 1,59 and 0,0 respectively.
  • the average refraction index (1.330), the average viscosity (1.0 mPa ⁇ s) and the dielectric constant (80,4) were set before the experiments.
  • quartz cuvettes were used for the analysis quartz cuvettes.
  • the containers were left to cool and the digested solutions were transferred in 25 ml flasks and filled to the volume indicated using purified water by means of a Milli-Q (Millipore, France) system.
  • the ICP-MS evaluations were carried out in a quadrupole XSERIES 2 ICP-MS system, (Thermo Fisher Scientific), operating at standard conditions.
  • the samples were placed in a concentrical quartz nebulizer by means of a peristaltic pump (selected speed: 30 rpm).
  • the concentration of the element was detected with respect to an external multielemental calibration standard of synthetic acid.
  • the entrapment efficiency was calculated using the following equation:
  • Entrapment efficiency (%) (Sb amount in polylactic acid nanocapsules)/ (Sb amount initially added to polylactic acid nanocapsules) x 100
  • the entrapment efficiency refers to the amount of Sb contained within the polylactic acid nanoparticles core.
  • the Turbiscan Lab ® is an optical analyzer that is able to identify the destabilizing phenomena in the samples to be analyzed by means of the multiple light diffusion (Celia et al., 2009; Celia et al., 2012).
  • the instrument is made of an intermittent light source with a wavelength in the near infrared (880 nm) and of two synchronized detectors (transmittance and reflected diffusion).
  • the emitted photon goes through the container with the sample in a cylindrical glass cell.
  • the transmittance detector (T) can detect the photon ( ⁇ ) that goes through the sample (at 0° with respect to the incident radiation), while the reflected diffusion detector (BS) detects the photon ( ⁇ ) diffused by the sample (at 135° with respect to the incident radiation).
  • the instrument was calibrated with a reflectance standard (polystyrene latex, totally opalescent) and with a transmittance standard (silicone oil, totally transparent).
  • the graphs obtained show the percentage of photons transmitted or reflected with respect to the reference standard. The analysis was carried out every minute for 1 hour.
  • ⁇ * was the average photon transport path in the dispersion analyzed. From a physical point if view, the value of ⁇ *( ⁇ , ⁇ ) in the dispersion analyzed was evaluated using the following equation:
  • ⁇ is the particle volume fraction
  • d is the average particle diameter
  • g(d) and QS(d) are the parameters given by the MIE equation to the Maxwell equation.
  • the data obtained were processed by means of the acquired data conversion software Easy soft and a Turbiscan Lab platform to obtain the kinetic stability profile of the sample as a function of the analysis timing.
  • the antimony release was evaluated following the dialysis method using Spectra/Por cellulose acetate dialysis tubings having a molecular cut-off of 12,000-14,000 daltons (Spectrum Laboratories Inc., The Netherlands) sealed at the ends with clips (Paolino et al., 2008).
  • the samples were, then, analyzed with the ICP-MS technique using the analytical procedures described above.
  • JTT4A.1 cells (murine macrophage cell line) were cultivated in culture plates (100 mmx20 mm) in an incubator at 37°C (5% CO2) using DMEM culture medium with glutamate, penicillin (100 Ul/ml), streptomycin (100 ⁇ g ml) and FBS (10% v/v). The medium was changed every 48 hours. When the cells were 80% confluent, they were treated with 2 ml trypsin, scraped from the plates and collected in a centrifuge tube containing 4 ml of culture medium. In addition, plates were washed with 2 ml of phosphate buffered saline solution (PBS) to remove the remaining cells, which were transferred to a centrifuge tube.
  • PBS phosphate buffered saline solution
  • Tubes were centrifuged at 1000 rpm at room temperature for 10 minutes with a Heraeus Sepatech Megafuge 1.0.
  • the cell pellet thus obtained was resuspended in a suitable volume of culture medium and seeded in culture plates before proceeding with the subsequent experiments.
  • the cytotoxic effects of the nanocapsules that do not contain the drug were evaluated by MTT test, or cell viability test, at different polymer concentration (from 0,01 to 100 uM) as previously described (Cosco et al., 2011). Briefly, the cultured cells were seeded into 96 well plates at a density of 5xl0 3 cells/0.2 ml and treated with the formulations to be examined for incubation times of 24, 48 e 72 hours. After each incubation time, a solution of tetrazolium salts was used to evaluate cell viability by means of their mitochondrial activity on said salts, with formation of intracellular blue-purple formazan crystals.
  • the interaction between the treated cells and the polylactic acid nanoparticles was evaluated by means of confocal laser scanning microscopy (CLSM).
  • CLSM confocal laser scanning microscopy
  • the monocytes were seeded (4 x 10 4 cells/ml) in 6 well plates where had been previously placed sterile microscopy glass slides, and incubated for 24 hours before treatment with polylactic acid nanocapsules containing DMPE-rhodamine, a fluorescent probe, to be analyzed at different contact times. After incubation, each well was washed three times with PBS to remove the surplus of particles in the suspension and the cells adhering to the slide were fixed using 1 ml of a 70% v/v ethanol solution.
  • each well was treated with 1 ml of a solution containing Hoechst (1/1000) for 30 minutes, to visualize at the microscope the cellular nuclei, and washed three times with 2 ml PBS. Finally, the plates were stored at the temperature of +4°C for the confocal microscopy analysis.
  • the slides, before the analysis, were placed onto slides using a 70% glycerol solution v/v, removing possible air bubbles, and then fixed with a transparent glue.
  • J774A.1 cells were seeded in Petri capsules and placed in an incubator for the whole night to allow them to adhere to the growth surface. After 24 hours, cells were treated with Glucantime ® or with polylactic acid nanocapsules containing MA at an Sb final concentration of 10 uM. The cells thus treated were washed and the amount of Sb contained in the culture medium was quantified by means of the above mentioned ICP-MS technique.
  • J774A.1 cells (4 ⁇ 10 4 cellule/ml) were seeded in 6 well plates and incubated for 24 day at 37°C in 5% CO2 to allow adhesion. In each well was previously placed a sterile slide. Subsequently, adherent macrophages were infected with L. tropica promastigote in a 10:1 (parasite/cell) ratio, and incubated for 4 hours at 37°C in 5% CO2. Free promastigotes were removed by washing the well with PBS. Glucantime and the polylactic acid nanocapsules containing the drug were tested in infected cells at different Sb doses (50 and 10 ⁇ ). After 48 hours, slides were fixed with methanol, dyed with 10% Giemsa, and examined using an optical oil immersion microscope.
  • mice were also used to study the biodistribution of Sb v contained in Glucantime ® and of the nanocapsules formulation, administering to each group one formulation or the other in the tail vein (100 ⁇ ), and determining the amount of antimony by means of the microwave digestion described above and ICP-MS measures (see “Antimonial Compound Entrapment Efficiency”).
  • organs were previously weighed (from 0.2 to 0.5 g) in fluoropolymer tubes and digested with7 ml HN03 (65% v/v, Merck) and 1 ml HC1 (34-37%, v/v, Carlo Erba), in the microwave digestion system.
  • the ANOVA method was used for the statistical analysis of the different experiments.
  • the Bonferroni t-test was used a posteriori to verify the ANOVA test. A p ⁇ 0.05 value was considered statistically significant. The values are reported as mean ⁇ standard deviation.
  • nanoparticles of the invention containing MA efficiently interact with macrophages, without damaging them or substantially altering their morphology and functions.
  • Pentoxifylline prevents the meglumine antimonate- induced renal toxicity in rats, but not that induced by the inorganic antimony pentachloride. Toxicology; 243(l-2):66-74, 2008.
  • Miele E Spinelli GP
  • Miele E Tomao F
  • Tomao S Albumin- bound formulation of paclitaxei (Abraxane ABI-007) in the treatment of breast cancer.

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Abstract

L'invention concerne des systèmes nanoparticulaires polymères comme véhicules efficaces de composés contenant de l'antimoine, ainsi que des substances contenant de l'antimoine comme l'antimoniate N-méthyl glucamine (Glucantime®) Plus particulièrement, le principe actif, utilisé pour le traitement de pathologies associées à une infection par Leishmania, a été piégé dans le « noyau » aqueux de nanocapsules ayant une couche externe, ou « coquille » d'acide polylactique (PLA), qui permettent de réduire la toxicité cellulaire et la modulation de sa biodistribution et de ses propriétés biopharmaceutiques, sans altérer son efficacité pharmacologique.
PCT/IT2015/000134 2014-05-19 2015-05-18 Systèmes nanoparticulaires visant à véhiculer des médicaments pour le traitement de pathologies liées à une infection par leishmania WO2015177820A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113616671A (zh) * 2020-05-09 2021-11-09 上海交通大学 微纳米MgH2化合物颗粒在抑制利什曼原虫感染及治疗利什曼病中的应用

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