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WO2025147516A1 - Compositions de nanoparticules lipidiques anti-inflammatoires et utilisations - Google Patents

Compositions de nanoparticules lipidiques anti-inflammatoires et utilisations Download PDF

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WO2025147516A1
WO2025147516A1 PCT/US2025/010090 US2025010090W WO2025147516A1 WO 2025147516 A1 WO2025147516 A1 WO 2025147516A1 US 2025010090 W US2025010090 W US 2025010090W WO 2025147516 A1 WO2025147516 A1 WO 2025147516A1
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lipid
composition
nanoparticle
nanoparticles
solution
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Bin Wu
Victoria Trantow
Sun ZHENGWANG
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Cytodigm Inc
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Cytodigm Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle

Definitions

  • Sialic acid also known as N-acetylneuraminic acid (or NANA), is a nine-carbon sugar that binds to sialic acid-binding immunoglobulin-like lectin (Siglec).
  • Sialic acid has mainly three derivatives: N-acetyl neuraminic acid (Neu5Ac), N-acetyl neuraminic acid hydroxyalkyl (Neu5Gc) and 3- deoxy-D-glycero-D-galacto-nonyl ketose (Kdn).
  • N-acetyl neuraminic acid Ne5Ac
  • N-acetyl neuraminic acid hydroxyalkyl Ne3
  • Kdn deoxy-D-glycero-D-galacto-nonyl ketose
  • Siglecs have an intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) that can mediate inhibitory signals upon binding to sialic acid and activate downstream inhibitory signaling through the recruitment of tyrosine phosphatases SHP-1 and SHP-2.
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • Sialic acid can also regulate the alternative pathway of complement activation.
  • Major serum protein complement factor H recognizes sialic acid as a “self’ marker, which helps to inhibit Clq/C3b fragment activation. Therefore, Sialic acid, when binding a Siglec receptor on immune cells or the complement factor H (CFH), serves as a self-associated molecular pattern (SMAP) to suppress over-reactive immune responses and maintain an overall homeostasis.
  • SMAP self-associated molecular pattern
  • the PLGA nanoparticles were then activated with a carbodiimide (EDC) followed by conjugation with a2,8 NANA to form the a2,8 NANA-NP.
  • EDC carbodiimide
  • this type of method suffers from several shortcomings: 1) the multi-step process is complex and difficult to scale up; 2) low conjugation efficiency due to steric hinderance which leads to low ligand density on NP surface and thus low binding efficiency; 3) introduction of harmful chemical residues in the pharmaceutical formulation that are difficult to remove; and 4) such conjugation may cause unwanted immunogenicity.
  • the present invention in part provides a lipid nanoparticle comprising an entity that contains one or multiple sialic acid (SA) moieties, the entity is preferably a ganglioside, a ganglioside derivative, a ganglioside mimetic, or a combination thereof.
  • SA sialic acid
  • the lipid nanoparticle of the present invention can effectively present SA moieties on the surface to bind Siglecs and complement factors to, inter alia, mitigate inflammation.
  • the present invention also provides methods of using said lipid nanoparticle compositions described herein for pharmaceutical applications.
  • the lipid nanoparticles provided herein are useful for treating inflammatory and autoimmune diseases.
  • Gangliosides are molecules composed of glycosphingolipids with one or more sialic acids linked on a sugar chain.
  • Preferred gangliosides include a ganglioside containing one sialic acid unit such as GM1, GM2, GM3, asialo-GMl, GAI, asialo-GM2, GA2, or two sialic acid units such as GDla, GDlb, GD2, and GD3, or three sialic acid units such as GTla, GTlb, GTlc, OAc-GTlb, GT3, or four salic acid units such as GQ1.
  • the anti-inflammatory lipid nanoparticle described herein comprises a ganglioside and one or more phospholipids.
  • the anti-inflammatory lipid nanoparticle described herein comprises a ganglioside, a mixture of cholesterol or cholesterol derivatives, and one or more phospholipids.
  • the lipid nanoparticle of the current invention is used to treat dry AMD such as geographic atrophy.
  • nanoparticles are preferably roughly round, sphere, or sphere-like in shape, and are generally within the size range of, e.g., between about 1-1,000 nm, between about 10-1,000 nm, or between about 50-1,000 nm, or between about 100-500 nm, as measured by laser diffraction, for example.
  • the subject nanoparticles may also include particles that are less likely to clump in vivo.
  • Particle size and size distribution can be measured by a dynamic light scattering instrument, e.g., a Malvern Zetasizer.
  • the particle size is typically reported as Z-average mean diameter.
  • Alternative techniques include, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation.
  • the term “nanoparticle” is not intended to convey any specific shape limitation. Such particles include, but are not limited to, those having a generally polyhedral or spherical geometry. Preferred particles are characterized by a spherical geometry typically produced by emulsion-based encapsulation processes.
  • nanoparticle encompasses both nanoparticle and microparticles.
  • a or “an” means one or more unless otherwise specified.
  • sialic acid residue and “sialic acid moiety” as well as their plural referents, and the like, are used interchangeably herein.
  • the term “subject” is used to mean an animal, preferably a mammal, including a human or non-human.
  • the terms “patient” and “subject” may be used herein interchangeably.
  • “Treatment” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing own or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease.
  • treatment includes to clinical intervention to alter the natural course of a disease in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology.
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • combinations of the invention are used to delay development of a disease or to slow the progression of a disease.
  • Gangliosides are molecules composed of glycosphingolipid with one or more sialic acids linked on the sugar chain. They form lipid rafts in the outer leaflet of the cell plasma membrane, especially in neuronal cells in the central nervous system. Gangliosides have been found to be highly important molecules in immunology as they participate in cellular proliferation, differentiation, adhesion, signal transduction, cell-to-cell interactions, tumorigenesis, and metastasis. More than 60 gangliosides are known.
  • Gangliosides can be named based on the number of sialic acid (SA) units they have in the molecule.
  • SA sialic acid
  • gangliosides having one SA unit are named “GM,” such as GM1, GM2, and GM3.
  • G stands for “ganglioside”
  • M stands for “mono.”
  • GD “GT” and “GQ” would refer to gangliosides having two (“di”), three (“tri”), and four (“quadruple”), respectively.
  • the structures of gangliosides GM1, GD3, GT lb, and GQ1 are shown above.
  • gangliosides contain sialic acid (SA) residues in their molecular structures.
  • SA units in the gangliosides can bind sialic acid-binding immunoglobulin-like lectins (Siglec) receptors expressed on various types of cells.
  • Siglec sialic acid-binding immunoglobulin-like lectins
  • many cell types including T cells, macrophages, microglial, neutrophils, mast cells, eosinophils, and basophils, all express one or multiple types of Siglecs on their surfaces, and such expressions are upregulated when inflammations occur.
  • Siglecs have an intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) that can mediate inhibitory signals upon binding to SA and activate downstream inhibitory signaling through the recruitment of tyrosine phosphatases SHP-1 and SHP-2.
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • SA can also regulate the alternative pathway of complement activation.
  • Major serum protein complement factor H recognizes sialic acid as a “self’ marker, which helps to inhibit Clq/C3b fragment activation. Therefore, Sialic acid, when binding a Siglec receptor on immune cells or the complement factor H (CFH), serves as a self-associated molecular pattern (SMAP) to suppress over-reactive immune responses and maintain overall homeostasis.
  • SMAP self-associated molecular pattern
  • lipid nanoparticles incorporating a ganglioside molecule on the nanoparticle surface can provide high avidity and efficiency for binding Siglecs and CFH to mitigate inflammation.
  • the ganglioside selected in making the lipid nanoparticles of the present invention may be a ganglioside containing one SA unit such as GM1, GM2, GM3, asialo-GMl, GAI, asialo-GM2, GA2, or two SA units such as GDla, GDlb, GD2 and GD3, or three SA units such as GTla, GTlb, GTlc, OAc-GTlb, GT3, or four SA units such as GQ1.
  • one SA unit such as GM1, GM2, GM3, asialo-GMl, GAI, asialo-GM2, GA2, or two SA units such as GDla, GDlb, GD2 and GD3, or three SA units such as GTla, GTlb, GTlc, OAc-GTlb, GT3, or four SA units such as GQ1.
  • gangliosides include ganglioside-total, Cl 8:0(2- NBD), GM1, NGcGM3, C18:0 GM3, C20:0 GM1, C17:0 GM1 and C18:0 GM1; commercially available at Avanti Polar Lipids, Birmingham, AL.
  • one sialic acid unit may be linked with its neighboring sialic acid unit via a2,3, a2,6, a2,8, or a2,9 linkage.
  • the ganglioside is selected from the group containing the gangliosides having one or two SA units in their molecular structures. In another embodiment, the ganglioside is selected from the group containing GM1, GM3, and GD3.
  • the amount of the ganglioside can vary depending on the number of components.
  • the gangliosides can be added to the formulation in an amount of at least about 0.1% molar percentage of the entire nanoparticle composition, preferably between 5-85%, or 20-75%, for example, in a two component system.
  • the ganglioside preferably has 1, 2, 3, 4 or more SA units.
  • the molar ratio of the ganglioside to total lipids can be between 0.1-50% or more, 0.5-20%, or 1-15% in a system having 3 or more components.
  • the lipid nanoparticle described herein further comprises a phospholipid or its derivative in addition to the ganglioside.
  • the phospholipid can be added in lieu of the cholesterol or in addition to it. In a two component system the phospholipid accounts for the remainder of the composition with the ganglioside.
  • PEG can also cause complement activation-related pseudo-allergies.
  • the immunogenicity mediated by PEG can prevent repeated dosing, thus hindering drug development.
  • the anti-PEG antibody may reduce the therapeutic effect of the API encapsulated in the LNP.
  • PEG-lipid in the LNP formulations.
  • PEG-replacing approaches use synthetic polymers similar to PEG and may thus also cause immunogenicity.
  • Certain natural or biocompatible substances proposed to replace the PEG-lipid such as hyaluronic acid and polysialic acid, must be chemically linked to lipids or LNPs.
  • these conjugates might induce an immune response, and the conjugation process can produce harmful byproducts.
  • some of these "PEG Substitutes" may be too large and hydrophilic, which could compromise the integrity and stability of the LNPs.
  • the addition of PEG-lipid is optional in the present invention as the ganglioside-containing LNP of the present invention can be made without the use of PEG-lipids.
  • the LNPs are free of or substantially free of PEG-lipids. “Substantially free” in this context is intended to mean a non-immunogenic amount and can preferably be less than 1%, such as less than 0.5% total weight.
  • the ganglioside in the lipid nanoparticles of the current invention can act to stabilize the nanoparticles without the presence of PEG. Gangliosides are naturally occurring and widely found in animals and humans. Therefore, gangliosides are safe and less likely to be immunogenic. Additionally, nanoparticles with an SA moiety may facilitate the RES (reticuloendothelial system) escape and render the nanoparticles prolonged circulation in the bloodstream.
  • RES reticuloendothelial system
  • the lipid nanoparticle further comprises one or more cationic or ionizable lipids.
  • a cationic lipid is a lipid having a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic (amino) lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.
  • the cationic lipid can be selected from, for example Dioleoyl- 3- trimethylammonium propane (DOTAP), l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), 3-(didodecylamino)-Nl,Nl,4-tridodecyl-l- piperazineethanamine (KL10), Nl-[2-(didodecylamino)ethyl]-Nl,N4,N4-tridodecyl-l,4- piperazinediethanami- ne (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy- N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]- di
  • An ionizable lipid is a class of lipid molecules that are neutral and non-ionic at physiological pH but will be protonated to become positively charged at lower pHs.
  • Examples of commercially available ionizable lipids include DLin- KC2-DMA, DLin-MC3- DMA, DLin-DMA, LP-01, DODMA, DODAP, ALC- 0315, SM-102, SS-OP, SS-EC, etc.
  • the ionizable lipid can be added to the formulation in an amount of at least about 0.1% molar weight percentage of total solids in the nanoparticle composition.
  • the molar ratio of the cationic or ionizable lipid can preferably be between 1-50%, 10-45%, or, more preferably between 15 and 40% of the total lipid composition.
  • the ionizable lipid can be added to the formulation in an amount of at least about 0.1% molar weight percentage of total solids in the nanoparticle composition.
  • the molar ratio of the cationic or ionizable lipid can preferably be between 1-75%, 10-60%, or, more preferably, between 20 and 50% of the total lipid composition.
  • the formulations include lipid nanoparticles that consist of a ganglioside (e.g., GD3, GM1, or GM3) and a phospholipid (e.g., a phosphatidyl choline or DSPC).
  • a ganglioside e.g., GD3, GM1, or GM3
  • a phospholipid e.g., a phosphatidyl choline or DSPC.
  • the molar ratio of such LNPs are preferably between 1 : 1 to 1 : 100, more preferably about 1 :2, molar ratio of ganglioside to phospholipid.
  • the formulations include lipid nanoparticles that consist of a ganglioside (e.g., GD3, GM3, or GM1) and cholesterol or a derivative thereof (preferably cholesterol).
  • the molar ratio of such LNPs are preferably between 1 : 1 to 1 : 100, more preferably about 1 :3, molar ratio of ganglioside to cholesterol or derivative.
  • the formulations include lipid nanoparticles that consist of a ganglioside (e.g., GD3, GM3, or GM1), a phospholipid (e.g., a phosphatidyl choline or DSPC) and cholesterol or a derivative thereof.
  • GM3, or GM1 a phospholipid (e.g., a phosphatidyl choline or DSPC), cholesterol or a derivative thereof, and an ionic lipid, such as MC3, and, optionally a PEG-lipid.
  • a phospholipid e.g., a phosphatidyl choline or DSPC
  • cholesterol or a derivative thereof e.g., a phosphatidyl choline or DSPC
  • an ionic lipid such as MC3, and, optionally a PEG-lipid.
  • the molar ratios of these multicomponent lipid systems can be characterized as a molar percentage of the total lipids in the formulation.
  • the ganglioside can be between 3% and 15% (5% to 10% is preferred) of the composition where the balance of the composition is phospholipid, ionic lipid, cholesterol and, optionally, PEG-lipid.
  • the molar amount of the ionic lipid is typically greater than the other components (individually), the molar amount of cholesterol (or a derivative thereof) is less than the ionic lipid but more than the phospholipid.
  • a PEG-lipid is added, it is typically a minor amount of the formulation, e.g., less than 5%, preferably less than 2%, of the total lipids.
  • the anti-inflammatory lipid nanoparticle described herein can optionally further comprise an active agent, such as a small molecule, a peptide, a protein, or a nucleic acid.
  • the active agent can be encapsulated within said lipid nanoparticle.
  • the amount of the active agent can be about 0.01 to about 50% (w/w) of the nanoparticle total solids, or about 0.05 to about 25%, about 0.1 to about 10%, about 0.2 to about 5%, about 0.5 to about 3%, about 1 to about 5%, or about 2 to about 5% (w/w) of the nanoparticle total solids.
  • the weight ratio of lipids to nucleic acid can be about 1 to 20, preferably 3 to 15, more preferably 4 to 10.
  • the concentration of nucleic acids to LNPs can be characterized by a N:P ratio.
  • N refers to the number of nitrogen atoms in the ionizable or cationic lipid (typically 1 nitrogen per molecule).
  • P refers to the number of phosphates in the nucleic acid molecule.
  • the nucleic acid molecule can be added to the lipid nanoparticle in a ratio preferably between 1 :2 to 10: 1, such as between 1 : 1 to 10: 1, more preferably between 3: 1 to 8: 1, such as 6: 1.
  • the active agent is advantageously an anionic drug (also referred to herein as an active pharmaceutical ingredient, or API).
  • active agents that are non- therapeutic, such as diagnostics can also be included as part of the particles according to the methods.
  • Preferred active ingredients are oligonucleotides, nucleic acid molecules and mimics thereof, such as DNA, RNA, PNA, siRNA, microRNA, circular RNA, antisense, oligonucleotide, aptamer, and a combination thereof.
  • API and “cargo” are used interchangeably herein.
  • the nanoparticles are free of active agents and, optionally, consist essentially of the lipid compositions described herein.
  • the nanoparticles are free of an active agent that is independently anti-inflammatory.
  • the lipid nanoparticle described herein can be manufactured by a coprecipitation process.
  • the ganglioside can be dissolved along with other lipids (cholesterol or phospholipid) in a preferably water-miscible organic solvent, such as an alcohol.
  • a preferably water-miscible organic solvent such as an alcohol.
  • the organic solution containing the ganglioside and other lipids is combined slowly (e.g., dropwise) or with mixing (e.g., via a microfluidic device) with an aqueous buffer solution to incur nanoprecipitation.
  • a small amount of organic solution can be added to the aqueous phase with mixing.
  • the particles may also be manufactured using a pre-assembled device such as a T- mixer, or an automated, microfluidic device such as NanoAssemblr Ignite of Cytivia and iNano L Series of Micro&Nano Biologies.
  • a pre-assembled device such as a T- mixer
  • an automated, microfluidic device such as NanoAssemblr Ignite of Cytivia and iNano L Series of Micro&Nano Biologies.
  • Exemplary solvents miscible with water include methanol, ethanol, isopropyl alcohol, acetone, tetrahydrofuran (THF), acetonitrile, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF).
  • cryoprotectants such as sucrose, lactose, glucose, and mannitol
  • said cryoprotectant is added to the LNP suspension at 1-30% (weight/volume) based on the total volume of the LNP suspension, more preferably at 5-15%.
  • the LNP suspension was diluted to 30 mL with distilled water, then transferred into a pre- hydrated Slide-A-LyzerTM dialysis cassette with a 20K MWCO and dialyzed against distilled water for 6 h.
  • the lipid nanoparticles were then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO.
  • the sample was collected from the upper filter and stored at -20° C in a 5% w/v sucrose solution.
  • the LNP suspension was diluted by adding 10 mL distilled water, then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO.
  • the sample was collected from the upper filter and stored at -20° C in a 5% w/v sucrose solution.
  • the nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z- average size was 102.2 nm with a poly dispersity of 0.35.
  • Each nanoparticle test article was tested first at a 1 : 10 dilution and then at a 2 nd dilution selected based on the response [nm] shift seen at the 1 : 10 dilution. These data were used to quantify the dissociation rate (kdiss) for each molecular binding interaction examined in the study.
  • the table below summarizes the Octet binding assay results for each nanoparticle analyte / protein ligand combination. Many of the exemplary lipid nanoparticles described herein displayed the desired strong binding to Siglecs and CFH, whereas the vehicle control LNP (Example 7) was measured to have no ligand binding. A very low kdiss indicates strong binding.
  • lipid nanoparticles described herein Compared to published data using different nanoparticle platforms, several of the exemplary lipid nanoparticles described herein exhibited superior binding affinities, up to 5 orders of magnitude stronger. In fact, some of the nanoparticle formulations tested herein bound Siglecs and CFH with such great affinity that they reached the Octet instrument’s limit of detection, with dissociate rates below 1.0 x 10' 7 . There is reason to believe that binding affinity could be clinically relevant and perhaps enable dosing regimens that are easier to adhere to. For example, this could alleviate the burden of frequent intravitreal injections as a dry-AMD treatment. The relationship between kdiss rates and the half-life of the binding interaction is shown below. If, as is suspected, decreased cellular and complement- mediated inflammation is effectuated by ample and durable binding of Siglecs and CFH, then these lipid nanoparticle formulations could promote persistent antiinflammatory effects, lasting on the order of months.
  • THP-1 monocytes were differentiated to Ml and M2 macrophages by culturing with 25 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours, and then adding either 20 ng/mL lipopolysaccharide (LPS) and 20 ng/mL interferon-gamma (IFN- y) for the Ml phenotype or 20 ng/mL IL-4 for the M2 phenotype.
  • PMA Phorbol 12-myristate 13-acetate
  • Nanoparticles tested in this assay included the following compositions: traditional LNP without ganglioside as vehicle control (NP7), GD3/Chol (NP2), MC3/Chol/DSPC/5%GD3 (NP5), and MC3/Chol/DSPC/10%GD3 (NP6).
  • NP7 traditional LNP without ganglioside as vehicle control
  • NP2 GD3/Chol
  • NP5 MC3/Chol/DSPC/5%GD3
  • NP6 MC3/Chol/DSPC/10%GD3
  • THP-1 monocytes to Ml macrophages were achieved with a protocol using PMA, LPS, and IFN-y.
  • THP-1 cells at 93% viability were counted and resuspended to 2 x 10 5 cells/mL in complete growth medium (RPML1640 base medium with 0.05 mM 2- mercaptoethanol, 10% fetal bovine serum (FBS), and IX penicillin/ streptomycin) supplemented with 25 nM PMA.
  • the cells were seeded in a 96-well tissue culture-treated plate at 4 x 10 4 cells per well and incubated (37 °C, 5% CO2) for 24 h.
  • a 5 mg/mL solution of the MTT reagent was prepared and filtered through a 0.22 pm syringe filter. Media was removed so that cells were in 100 pL/well. 10 pL of the MTT solution was added to each well, and the plate was incubated for 4 hours. Black formazan crystals were visible in positive control and experimental wells, not negative control conditions. 100 pL of isopropanol with 0.04 N HC1 was added per well and pipette-mixed until formazan crystals dissolved. A VarioskanTM LUX multimode microplate reader was used to measure absorbance at 570 nm and a reference wavelength of 630 nm. The MTT assay and viability results were shown in Figure 2 and Figure 3.
  • THP-1 monocytes were differentiated to Ml and M2 macrophages by culturing with 25 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours, and then adding either 20 ng/mL lipopolysaccharide (LPS) or 20 ng/mL interferon-gamma (IFN- y) for the Ml phenotype or 20 ng/mL IL-4 for the M2 phenotype. After 48 hours, differentiated Ml macrophages were washed and replaced with fresh media containing the differentiation cytokines and lipid nanoparticle treatments. The Ml macrophages were incubated for 24 h with 0.
  • PMA Phorbol 12-myristate 13-acetate
  • nanoparticles tested in this essay were GD3/DSPC prepared by PPT (NP 1.2). As shown in Figure 4, all nanoparticles tested demonstrated the ability to reduce the concentrations of IL-6.
  • THP-1 monocytes were differentiated to Ml and M2 macrophages by culturing with 25 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours, and then adding either 20 ng/mL lipopolysaccharide (LPS) or 20 ng/mL interferon-gamma (IFN- y) for the Ml phenotype or 20 ng/mL IL-4 for the M2 phenotype. After 48 hours, differentiated Ml macrophages were washed and replaced with fresh media containing the differentiation cytokines and lipid nanoparticle treatments. The Ml macrophages were incubated for 24 h with 0.
  • PMA Phorbol 12-myristate 13-acetate
  • nanoparticles tested in this assay included the following two compositions: GD3/Chol prepared by PPT (NP 2.2) and GD3/Chol prepared by iNano L+ (NP2.3).
  • IL-6 IL-6
  • 5B NNF-a
  • 5C IL-ip

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Abstract

La présente invention concerne une nanoparticule lipidique comprenant une entité qui contient une ou plusieurs fractions d'acide sialique (AS), l'entité est de préférence un ganglioside, un dérivé de ganglioside, un mimétique de ganglioside, ou une combinaison de ceux-ci. La nanoparticule lipidique de la présente invention peut présenter efficacement des fractions d'AS sur la surface pour lier des Siglecs et des facteurs du complément pour, entre autres, atténuer l'inflammation. La présente invention concerne également des procédés d'utilisation desdites compositions de nanoparticules lipidiques présentement décrites pour des applications pharmaceutiques. Par exemple, les nanoparticules lipidiques de l'invention sont utiles pour le traitement de maladies inflammatoires et auto-immunes.
PCT/US2025/010090 2024-01-03 2025-01-02 Compositions de nanoparticules lipidiques anti-inflammatoires et utilisations Pending WO2025147516A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210128489A1 (en) * 2014-04-16 2021-05-06 Trustees Of Boston University Gm3 functionalized nanoparticles
US20220142935A1 (en) * 2020-10-05 2022-05-12 Phosphorex, Inc. Pharmaceutical composition of siglec-binding agents

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210128489A1 (en) * 2014-04-16 2021-05-06 Trustees Of Boston University Gm3 functionalized nanoparticles
US20220142935A1 (en) * 2020-10-05 2022-05-12 Phosphorex, Inc. Pharmaceutical composition of siglec-binding agents

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