EP4392021A1 - Liposomen, zusammensetzungen damit, verwendungen davon - Google Patents

Liposomen, zusammensetzungen damit, verwendungen davon

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Publication number
EP4392021A1
EP4392021A1 EP22769372.8A EP22769372A EP4392021A1 EP 4392021 A1 EP4392021 A1 EP 4392021A1 EP 22769372 A EP22769372 A EP 22769372A EP 4392021 A1 EP4392021 A1 EP 4392021A1
Authority
EP
European Patent Office
Prior art keywords
liposomes
phospholipid
solution
imatinib
obtaining
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
EP22769372.8A
Other languages
English (en)
French (fr)
Inventor
Silvia ARPICCO
Barbara Rolando
Barbara Stella
Federica MELONI
Laura PANDOLFI
Emanuela COVA
Veronica CODULLO
Rosanna DI PAOLA
Salvatore Cuzzocrea
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fondazione Irccs Policlinico San Matteo
Universita degli Studi di Torino
Original Assignee
Fondazione Irccs Policlinico San Matteo
Universita degli Studi di Torino
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 Fondazione Irccs Policlinico San Matteo, Universita degli Studi di Torino filed Critical Fondazione Irccs Policlinico San Matteo
Publication of EP4392021A1 publication Critical patent/EP4392021A1/de
Pending legal-status Critical Current

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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • 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
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • FIG. 1 Cell viability results after incubation of primary myofibroblast lines (LFs) derived from patients with chronic lung rejection (CLAD;A) , fibrosis- associated collagen disease (ILD-CTD;B) or immortalised human lung carcinoma cell line (A549;C) with imatinib- containing liposomes (LIPIm) , HA-coated liposomes containing imatinib (LIP-HAIm) and imatibin (Im) alone, at the same imatinib concentration (30 pM) after 24, 48 and 72 h. Data are represented as mean ⁇ standard deviation of the percentage compared to the control set at 100%. Statistical analysis: **, p ⁇ 0.01 vs. CTR; *, p ⁇ 0.05 vs . CTR .
  • FIG. 1 Results of western blot protein quantification performed on the cell lysate of CLAD- and ILD-CTD-derived LFs after treatment for 24 h with LIPIm, LIP-HAIm and imatinib (Im) .
  • CTR Quantification of phosphorylated cAbl normalized to control
  • CTR quantification of type I collagen normalized to control
  • Hemocytometer cell count results in bronchoalveolar lavage fluid in the presence of trypan blue dye (A) .
  • Hemocytometer count results of neutrophils (B) , macrophages (C) and lymphocytes (D) in the presence of Wright-Giemsa dye in bronchoalveolar lavage fluid in mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) .
  • FIG. 6 Representative images acquired with the light microscope of lung tissue sections stained with hematoxylin and eosin from mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposome with imatinib and hyaluronic acid intratracheally (LIPO-IM-HA) , Aschroft score (A) indicates histological damage.
  • Sham saline
  • CTR bleomycin and saline
  • HEPES bleomycin and HEPES
  • IM IT bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM-HA Aschroft score
  • Figure 7 Representative images acquired with the light microscope of Masson's trichrome-stained lung tissue sections of mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) , collagen quantitation (A) .
  • Sham saline
  • CTR bleomycin and saline
  • HEPES bleomycin and HEPES
  • IM IT bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM-HA bleomycin and liposomes with imatinib and hyaluronic acid
  • FIG 8. Representative images acquired with the light microscope of the lung tissue sections on which an immunohistochemical analysis was performed with the TGF- lp antibody of the mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) , quantification of TGF-lp expression (A) .
  • Sham saline
  • CTR bleomycin and saline
  • HEPES bleomycin and HEPES
  • IM IT bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM-HA quantification of TGF-lp expression
  • Figure 9 Representative images acquired with the light microscope of the lung tissue sections on which immunohistochemical analysis was performed with the CD4 antibody from mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM-HA) , quantification of CD4 expression (A) .
  • Sham saline
  • CTR bleomycin and saline
  • IM IT bleomycin and HEPES bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM-HA bleomycin and liposomes with imatinib and hyaluronic acid
  • Figure 10 Representative images acquired with the light microscope of lung tissue sections on which immunohistochemical analysis was performed with the CD8 antibody of mice treated with saline (Sham) , bleomycin and saline (CTR) , bleomycin and HEPES (HEPES) , bleomycin and imatinib intratracheally (IM IT) , bleomycin and imatinib intraperitoneally (IM IP) , bleomycin and liposomes with imatinib and hyaluronic acid (LIPO-IM- HA) , quantification of CD8 expression (A) .
  • Sham saline
  • CTR bleomycin and saline
  • HEPES bleomycin and HEPES
  • IM IT bleomycin and imatinib intratracheally
  • IM IP bleomycin and imatinib intraperitoneally
  • LIPO-IM- HA quantification of CD8 expression
  • This disclosure relates to a liposomal nanovehicle functionali zed on the surface with hyaluronic acid (HA) in order to facilitate the selective delivery of an active ingredient towards diseased cells and alveolar macrophages present in chronic and severe respiratory diseases .
  • HA hyaluronic acid
  • Liposomes are phospholipid vesicles that have a membrane , the liposomal membrane , comprising a lipid bilayer, general ly a phospholipid bilayer . Liposomes have nanometric dimensions , enclose an aqueous core and may be used in the clinic for the delivery of active ingredients . They can encapsulate both hydrophilic and lipophilic molecules , allowing the gradual release of the active ingredient , thus modi fying its pharmacokinetic profile .
  • the liposomes obj ect of the present disclosure are HA-coated liposomes having a molecular weight higher than 17000 Daltons , preferably between 20000 and 60000 Daltons .
  • HA is directly bound to the head of a phospholipid of the lipid bilayer of the liposomal membrane .
  • the HA is directly bound to the head of a membrane phospholipid comprising an amine group .
  • the HA is directly bound to an amine group of a membrane phospholipid .
  • the weight ratio of hyaluronic acid to membrane phospholipids is between 1 and 5 .
  • the phospholipid bilayer of the liposomal membrane may also comprise cholesterol .
  • the phospholipid of the phospholipid bilayer of the liposomal membrane may be selected from 1 , 2-dipalmitoyl- sn-glycero-3-phosphoethanolamine ( DPPE ) , phosphatidylglycerol ( PG) , phosphatidylcholine ( PC ) and combinations thereof .
  • DPPE 2-dipalmitoyl- sn-glycero-3-phosphoethanolamine
  • PG phosphatidylglycerol
  • PC phosphatidylcholine
  • PC phosphatidylcholine
  • the coated liposomes obj ect of the present disclosure may have an average diameter ranging from 100 nm to 300 nm, preferably equal to 250 nm .
  • the mean diameter value was analyzed by the quasi-elastic light scattering ( QELS ) technique at 25 ° C .
  • the coated liposomes have a Z potential value ranging from - 10 mV to -50 mV, preferably equal to -33 mV .
  • the liposomes obj ect of the present disclosure may contain at least one active ingredient . From the electron microscope analysis it was also observed that the liposomes have a spherical shape and the presence of the precipitated active ingredient inside them was confirmed .
  • the liposomes retain approximately 95% of the initial active ingredient content , in particular imatinib, after storage at 4 ° C for 3 weeks . During this period, no appreciable changes in diameter and Z-potential , nor precipitation or aggregation phenomena are observed .
  • the encapsulation method carried out by the Inventors and described below provides a series of advantages , among which it allows to obtain stable formulations : the Inventors have in fact observed that 20% of imatinib, as an active ingredient , is released in 24 hours in HEPES buf fer and 30% in 72 hours ; in serum the release is about 50% in 24 hours and 80% in 72 hours .
  • the liposomes obj ect of the present disclosure as well as the compositions containing them may be used to deliver the active ingredients contained therein to speci fic targets and may also be administered by inhalation .
  • the diseases which may be ef fectively treated with the liposomes obj ect of the present disclosure are respiratory diseases , preferably pulmonary fibrotic diseases .
  • treatable diseases may be selected from idiopathic pulmonary fibros is , pulmonary fibrosis associated with collagen diseases and bronchiolitis obliterans .
  • Bronchiolitis obliterans may result from lung transplantation or chronic mani festation after allogeneic bone marrow stem cell transplantation .
  • the liposomes obj ect of the present disclosure coated with HA having a speci fic molecular weight represent an absolute innovation and an important step forward in the treatment of these serious diseases , with a considerable economic and social impact .
  • Bronchiolitis obliterans which represents the failure of a lung transplant or the expression of chronic pulmonary graft , is associated with high costs to the healthcare system, requiring frequent evaluations and hospitali zations , costly therapies and complex support in the phase of disease progression, while pulmonary fibrosis is often the cause of severe disability and death in patients .
  • pulmonary fibrosis is often the cause of severe disability and death in patients .
  • idiopathic form it is not associated with other systemic diseases
  • systemic sclerosis or autoimmunebased inflammatory myopathy it is often unresponsive to the immunosuppressive therapy adopted for these pictures .
  • mesenchymal cell growth factors such as PDGF, TGF-beta and VEGF
  • the action of these factors is mediated by several protein kinases that mediate apoptosis resistance , inhibition of LFs proli feration and extracellular matrix production .
  • TK tyrosine kinase
  • imatinib which not only inhibits BCR-ABL, but is equally potent against other receptors with tyrosine kinase properties , such as PDGFRa and c-KIT .
  • nintedanib Another compound, already widely used in the treatment of pulmonary fibrosis , both idiopathic and associated with collagen diseases , and administered orally, is nintedanib, which is able to inhibit PDGF a and B , FGF 1-3 and EGFR 1-3 receptor-associated TK .
  • Liposomal formulations containing anti fungal or anticancer drugs are commercially available for intravenous administration .
  • liposomal amikacin which is indicated in the treatment of mycobacterial pulmonary diseases refractory to first- line antibiotic therapy
  • liposomal cyclosporine under registration for bronchiolitis obliterans
  • Commercially available formulations are not functionali zed and therefore do not have a speci fic cellular target . Therefore , to date , there is no liposomal formulation with active targeting capability in advanced development that may also be administered by inhalation .
  • the local administration of active ingredients via the liposomes described here is extremely ef fective in that it allows the active ingredients to speci fically reach the cells most involved in the pathogenic process , i . e . cells expressing the CD44 receptor ( LFs , inflammatory cells , activated endothelial cells ) .
  • the salt contained in the buf fer solution of lipid film hydration step may be selected from sodium citrate , sodium phosphate , sodium sulphate , sodium acetate , ammonium citrate , ammonium phosphate , ammonium sulphate and ammonium acetate .
  • the pH of the buf fer solution comprising a salt preferably sodium citrate , may be between 4 and 5 .
  • the Inventors have observed that optimal results in terms of encapsulation yield of the active ingredient are obtained when the salt contained in the buf fer solution is sodium citrate .
  • the pH of the buf fer solution comprising sodium citrate is 4 . 38 .
  • the method does not involve the use of ammonium sulphate .
  • the pH change step may be carried out by subjecting the suspension to gel filtration on a packed column, for example with Sepharose CL-4B, eluting with buffer, preferably HEPES, pH 7.4.
  • buffer preferably HEPES, pH 7.4.
  • the liposomal suspension added with the active ingredient may be kept for a period of time comprised between 20 minutes and 60 minutes, preferably equal to 30 minutes, at a temperature comprised between 30°C and 40°C, preferably equal to 37°C.
  • the phospholipid comprising an amine group is added solubilized in the solvent, preferably comprising chloroform and methanol.
  • the mixture may comprise chloroform and methanol in a 1:1 weight ratio.
  • the phospholipid may be added to the solvent in a concentration between 0.02 mmol and 0.1 mmol.
  • the solution may be subjected to purification and optionally washed.
  • the purification may be carried out by dialysis.
  • the washing may be carried out for example with DCM.
  • the solution may then be subjected to a lyophilization step to obtain the lyophilized HA-f conjugate.
  • the liposomes obtained with the described method may have an average diameter between 100 nm and 300 nm, when determined using the quasi-elastic light scattering (QELS) technique.
  • QELS quasi-elastic light scattering
  • the diameter is preferably about 200 nm; in the case of HA-coated liposomes, the diameter is preferably about 250 nm.
  • the presence of HA on the surface of the liposomes therefore leads to an increase in the value of the average particle diameter.
  • the Z-potential value may be around -30 mV for uncoated liposomes and slightly more negative, around -33 mV, for HA-coated liposomes, due to the presence of the negatively charged carboxyl groups of HA.
  • a composition comprising 1 , 2-distearoyl-sn- glycerol-3-phosphocholine (DSPC) , cholesterol (COL) and phosphatidylglycerol (PG) in a molar ratio of 70:30:3 was used to prepare the liposomes.
  • DSPC 2-distearoyl-sn- glycerol-3-phosphocholine
  • COL cholesterol
  • PG phosphatidylglycerol
  • the components were solubilized in chloroform and then the lipid film was prepared by evaporating the solvent under reduced pressure in the Rotavapor® and then under vacuum.
  • the lipid film was hydrated with citrate buffer (solution of citric acid and sodium citrate, pH 4.38) , sonicated and vortexed.
  • the liposomes were extruded under nitrogen pressure through polycarbonate filters (pore diameter 400 and 200 nm) using an extruder (Extruder; Lipex, Vancouver, Canada) at a temperature of 40°C. They were then subjected to gel filtration on a column packed with Sepharose CL-4B eluting with HEPES buffer pH 7.4 to change the pH of the liposomal suspension.
  • a solution of imatinib mesylate in HEPES buffer (0.5 mg/100 pl) was then added to the liposomal suspension and incubated for 30 minutes at 37°C; the liposomal preparation was then purified on a packed column with Sepharose CL-4B eluting with HEPES buffer pH 7.4, removing the unencapsulated active ingredient by gel filtration.
  • the same procedure was used to obtain the HA-coated liposomes by replacing the PG with the HA-f conjugate.
  • the HA-f conjugate was obtained by conjugating HA of molecular weight 44000 Da with the phospholipid 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) .
  • DPPE dipalmitoyl-sn-glycero-3-phosphoethanolamine
  • the HA-f conjugate was obtained by a reductive amination reaction between HA and a phospholipid containing an amino group (PE) .
  • PE phospholipid containing an amino group
  • HA with a molecular weight of 44000 Da was solubilized in a mixture of DCM and DMSO (1:1) in a sonicator bath for 90 minutes.
  • the liposomal preparations were stored at a temperature of 4 °C.
  • the average diameter and poly-dispersion index of the liposomes were determined at 25°C by means of the quasi-elastic light scattering (QELS) technique, and the surface charge was assessed by Z-potential determination by performing triplicate measurements.
  • the morphology of the liposomes was determined by cryo-TEM (cryogenic- Transmission Electron Microscopy) analysis.
  • the poly- dispersion index was less than 0.2 for the uncoated liposomes and the HA-coated liposomes object of this disclosure .
  • the cells used express high levels of the CD44 protein, as described in the scientific article Antibody-engineered nanoparticles selectively inhibit mesenchymal cells isolated from patients with chronic lung allograft dysfunction. Nanomedicine (Lond) . (2015) 10: 9-23.
  • the Inventors evaluated the effect of liposomes containing imatinib as active ingredient on cell viability using both A549 and LFs derived from CLAD and ILD-CTD.
  • the viability assay was carried out by MTT assay, which gives the possibility to quantify cell viability by means of mitochondrial activity.
  • 3 x 10 3 A549 cells or LFs were plated and incubated with LIP- HAIm, LIPIm, imatinib by incubating at the same concentration of imatinib (30 uM) for up to 72 h.
  • the solubility of imatinib in citrate buffer and ammonium sulphate solution was determined by preparing a suspension of the active ingredient in each of the two media, incubating it at 25°C for 24 hours and analyzing the filtrate via HPLC .
  • the peculiar characteristic of the liposomes disclosed here is that they act preferentially on fibroblasts and inflammatory cells (in particular macrophages and lymphocytes expressing CD44) , which are the main effectors (key players) of chronic progressive fibrosing diseases. On myofibroblasts, in particular, they act by inhibiting their proliferation, migration and extracellular matrix deposition. In vivo efficacy test
  • mice Male GDI mice (25-30 g, Envigo, Milan, Italy) were housed in a controlled location. They received food and water ad libitum. The study was approved by the Animal Care Review Board (OPBA) of the University of Messina. All in vivo experiments followed the new US, European, Italian and ARRIVE guidelines.
  • OPBA Animal Care Review Board
  • mice received bleomycin administration and were treated daily with the vehicle (saline solution (50 pL) ) ;
  • mice received bleomycin administration and were treated intratracheally with hepes (50 pL) ;
  • mice received bleomycin administration and were treated intratracheally with Imatinib (50 pL of 150 pg/ml) ;
  • mice received bleomycin administration and were treated daily with imatinib (10 mg/ml) intraperitoneally (IP) ;
  • LIPO-IM-HA mice received bleomycin administration and were treated daily with liposome with hyaluronic acid-functionalized imatinib (LIPO-IM-HA) (50 pL of 150 pg/ml) ;
  • mice were euthanized 28 days after bleomycin instillation and tissues were collected for lesion analysis .
  • Survival rate and body weight gain mortality and body weight were assessed daily up to 28 days.
  • Bronchoalveolar lavage (BAL) : twenty-eight days after bleomycin instillation, the mice were sacrificed and the tracheas were cannulated to perform the lavage. A total of 0.5 ml of phosphate-buff ered saline (PBS) was used. The recovered BAL fluid was centrifuged, the pelleted cells were harvested and the supernatants were stored at -20°C. In the presence of trypan blue dye, total BAL cells were counted using a hemocytometer. For a differential leucocyte count, the bronchoalveolar lavage (BAL) wash was stained with Wright-Giemsa. After staining, the differential count was performed using the standard morphological protocol under the light microscope .
  • PBS phosphate-buff ered saline
  • MPO activity was defined as the amount of enzyme that degrades 1 pmol peroxide per minute at 37 °C and was expressed in units per gram of wet tissue weight.
  • the pellets were resuspended in a second buffer containing 150 mM sodium chloride (NaCl) , 1% Triton X- 100, 1 mM ethylene glycol tetraacetic acid (EGTA) , 10 mM tris-chloric acid (HC1) pH 7.4, 0.2 mM PMSF, 1 mM ethylenediaminetetraacetic acid (EDTA) , 0.2 mM sodium orthovanadate and 20 pm leupeptin. After centrifugation at 4°C and 15000 g for 30 min, the nuclear proteins containing the supernatants were stored at -80°C for further analysis.
  • NaCl sodium chloride
  • HC1 tris-chloric acid
  • PMSF 1 mM ethylenediaminetetraacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • anti-IkB- alpha (1:1000, Santa Cruz Biotechnology) or anti-NF-kB p65 (1:1000; Santa Cruz Biotechnology) or anti-p-p38 (1:1000, Santa Cruz Biotechnology) or anti-p-JNK (1:1000, Santa Cruz Biotechnology) or anti-MMP2 (1:1000, Santa Cruz Biotechnology) or anti-MMP9 (1:1000, Santa Cruz Biotechnology) or anti-TRAF-6 (1:1000, Santa Cruz Biotechnology) in IxPBS, 5% w/v skimmed milk powder and 0.1% Tween-20 and incubated at 4 °C, overnight.
  • the membranes were incubated with peroxidase-conj ugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research) for 1 hour at room temperature. To verify that the membranes were loaded with equal amounts of protein, they were also incubated with antibody against laminin (1:1000; Santa Cruz Biotechnology) and beta-actin (1:1000; Santa Cruz Biotechnology) . The signals were detected with the enhanced chemiluminescence detection system reagent according to the manufacturer's instructions (SuperSignal West Pico Chemiluminescent Substrate, Pierce) .
  • Immunohistochemical analysis the antibodies that were incubated O/N on the brain sections were anti-TGF- beta (Millipore, 1:500 in PBS, v/v, AB152, Burlington, MA, USA) , anti-CD4 (Santa Cruz Biotechnology, 1:300 in PBS, v/v, 65G10 sc-32258, Dallas, TX, USA) and anti-CD8 (Santa Cruz Biotechnology, 1:50 in PBS, v/v, LB509 sc- 58480, Dallas, TX, USA) .
  • TGF- beta Millipore, 1:500 in PBS, v/v, AB152, Burlington, MA, USA
  • anti-CD4 Santa Cruz Biotechnology, 1:300 in PBS, v/v, 65G10 sc-32258, Dallas, TX, USA
  • anti-CD8 Santa Cruz Biotechnology, 1:50 in PBS, v/v, LB509 sc- 58
  • the Image J IHC profiler plug-in was used for densitometric analysis. When this is selected, it automatically draws a histogram profile of the deconstructed DAB image and a corresponding score log is shown.
  • the histogram profile corresponds to the positive pixel intensity value obtained by the computer program.
  • mice Twenty-eight days after bleomycin instillation, the HEPES-treated mice showed recruitment of inflammatory cells in the bronchoalveolar lavage fluid, compared to the sham group (Figure 5A) .
  • neutrophils Figure 5B
  • macrophages Figure 5C
  • lymphocytes Figure 5D
  • LIPO-IM-HA significantly reduced the infiltration of inflammatory cells into the bronchoalveolar lavage fluid.
  • - hydrating the lipid film in a solution containing at least one salt preferably selected from sodium salt , potassium salt , ammonium salt and obtain a second solution, subj ecting the second solution to extrusion and obtaining a suspension comprising liposomes .
  • extrusion may be conducted using an extruder, preferably at a temperature between 20 ° C and 60 ° C, more preferably 40 ° C .
  • the method for producing liposomes may provide that HA, preferably having a molecular weight greater than 17000 Da, bound to at least one phospholipid is added in the step of hydrating the lipid film .
  • HA bound to at least one phospholipid may be obtained by the steps of :

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Dispersion Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pulmonology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Otolaryngology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
EP22769372.8A 2021-08-24 2022-08-23 Liposomen, zusammensetzungen damit, verwendungen davon Pending EP4392021A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT102021000022253A IT202100022253A1 (it) 2021-08-24 2021-08-24 Liposomi, composizioni che li comprendono, relativi usi
PCT/IB2022/057879 WO2023026179A1 (en) 2021-08-24 2022-08-23 Liposomes, compositions comprising the same, uses thereof

Publications (1)

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EP4392021A1 true EP4392021A1 (de) 2024-07-03

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IT (1) IT202100022253A1 (de)
WO (1) WO2023026179A1 (de)

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Publication number Priority date Publication date Assignee Title
DK2706988T3 (da) * 2011-05-12 2020-01-20 Yissum Res Dev Co Of Hebrew Univ Jerusalem Ltd Liposomer omfattende polymer konjugerede lipider og relateret brug

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IT202100022253A1 (it) 2023-02-24

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