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WO1999054344A1 - Composition comprenant une combinaison de liposomes cationiques derives et d'agent actif - Google Patents

Composition comprenant une combinaison de liposomes cationiques derives et d'agent actif Download PDF

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Publication number
WO1999054344A1
WO1999054344A1 PCT/GB1999/001171 GB9901171W WO9954344A1 WO 1999054344 A1 WO1999054344 A1 WO 1999054344A1 GB 9901171 W GB9901171 W GB 9901171W WO 9954344 A1 WO9954344 A1 WO 9954344A1
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Prior art keywords
cationic liposome
achx
plasmid
liposomes
cells
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English (en)
Inventor
Andrew David Miller
Christopher John Etheridge
Raymond Fellowes
Patricia Woo
Arno Tido Duffels
Steven Victor Ley
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Ip2ipo Innovations Ltd
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Imperial College Innovations Ltd
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Priority to AU36141/99A priority Critical patent/AU3614199A/en
Publication of WO1999054344A1 publication Critical patent/WO1999054344A1/fr
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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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers

Definitions

  • the present invention relates to a compound, a cationic liposome comprising the compound, and to the use of that compound in therapy, in particular gene therapy (especially gene transfer). 5
  • EP-B-0 452 457 to Vanderbilt University discloses uses of a cationic liposome to transfect DNA into a cell in a target organ. However, the site of administration must be proximal to the target organ. 5
  • cationic liposomes performed poorly in comparison to adenoviral and retroviral vectors.
  • a particular problem with cationic liposomes as a drug delivery system is their non-specific adherence to many cell types.
  • Rheumatoid arthritis is generally considered to be an autoimmune disease and is characterised by inflammation of the synovial joints, subsequent destruction of the synovial lining and erosion of the underlying bone.
  • Current treatments involve the use of steroid anti-inflammatory agents and gold containing compounds.
  • high doses of such agents must be given. Such high doses are associated with side effects. 2
  • cationic liposomes can be used to administer agents of interest to a site which does not have to be proximal to the site of administration. Further since the invention provides a targeted drug delivery system it may bring about diminished toxic side effects and an increase in pharmacological activity.
  • a combination of a derivatised cationic liposome and an agent of interest in the preparation of a composition for delivering the agent of interest to a site that is distal to the site of administration of the composition, wherein the derivatised cationic liposome comprises a cationic liposome forming entity and a head group and/or a ligand, and wherein the head group and/or ligand increases macrophage uptake of the cationic liposome.
  • derivatised we include derivatising existing or newly formed cationic liposomes and also the formation of derivatised cationic liposomes ab initio.
  • cationic liposome we mean a combination of chemical species that are capable of forming a liposomal structure having one or more cationic groups giving a net positive charge.
  • liposomes are made from surface active agents which spontaneously form closed spherical structures when dispersed in water. Liposomes can describe unilamellar vesicles, i.e. where surface active agents are organised in one spherical bilayer. Additionally, the number of bilayers wrapped concentrically around can be greater than one. When there is more than one bilayer, typically tens of bilayers, it is commonly known as an oligolamellar vesicle.
  • the surface active agents are lipids.
  • lipid we mean a 3 natural or synthetic substance which is soluble in hydrocarbon solvents and which has a nonpolar "tail” and a highly polar "head” group. In aqueous solution the nonpolar tails cluster together leaving the polar head groups exposed to the aqueous environment.
  • capable of forming a liposomal structure is intended to include lipids or other surface active agents which (a) can by themselves form spontaneously into bilayer vesicles in water or (b) can be stably incorporated into the bilayer formed by molecule(s) of type (a).
  • Cationic liposomes are liposomes that comprise lipids or other surface active agents which have one or more cationic moieties with a net positive charge. There may be more than one type of cationic moiety. There may also be neutral lipids or other such neutral surface active agents included.
  • cationic liposome forming entity lipids or other surface active agents which are capable of forming a cationic liposome with or without an included ligand, and lipids or other surface active agents which require the added ligand to be capable of forming a cationic liposome.
  • ligand we mean not only entities which are linked to the cationic liposome forming entity but also entities which are in admixture with the cationic liposome forming entity.
  • the cationic liposome forming entity may be linked to or be in admixture with more than one ligand which may be the same or different.
  • the ligand may be linked to the cationic liposome forming entity by a bond, such as a covalent or hydrogen bond.
  • the ligand may be linked directly to the cationic liposome forming entity or via a spacer group. Indeed the spacer group may also be a ligand, i.e. a ligand may be linked indirectly to the cationic liposome forming entity via another ligand.
  • the ligand may be linked to the external surface of the liposome and/or the internal surface of the liposome.
  • the head group may also be 4 linked to the cationic liposome forming entity via a spacer group.
  • agent of interest we mean the agent which is to be delivered to a site distal to the site of administration.
  • the agent of interest may be a drug (including a pro-drug), a nucleotide sequence and/or expression product thereof, a marker, protein and/or peptide.
  • nucleotide sequence includes DNA, RNA and antisense sequences.
  • nucleotide sequence also includes part or all of an expression system.
  • macrophage uptake is better than macrophage uptake of 3 ⁇ -[N(7V'N'-dimethylaminoethane)carbamoyl]cholesterol (commonly known as "DC- Chol", the structure of DC-Choi is shown in Figure 3).
  • a head group or ligand entity increases macrophage uptake may be tested against a control using cell staining or in situ hybridisation techniques. Examples of these techniques are described in Example 1 below.
  • distal we mean that the following administration of the composition the agent of interest is delivered to a site in the body outside of the first capillary bed and/or organ, tissue or cell adjacent the administration site. Included within the term “adjacent" are sites which are downstream from an injection.
  • the site to which the agent of interest is delivered may be an organ, tissue or cell.
  • a combination of a derivatised cationic liposome and an agent of interest in the preparation of a composition for delivering the agent of interest to an inflamed site, wherein the derivatised cationic liposome comprises a cationic liposome forming 5 entity and a head group and/or a ligand, and wherein the head group and/or ligand increases macrophage uptake of the cationic liposome.
  • liposome mediated delivery is a viable method for the delivery of an agent of interest to a distal site and/or an inflamed site.
  • the inflamed site may result from the disease rheumatoid arthritis.
  • the present invention shows that liposome mediated gene delivery is a viable method for the delivery of therapeutic genes or the expression product thereof to distant inflammatory sites and that the transgene is expressed in sufficient quantities to have a beneficial effect on established arthritis.
  • the present invention provides amelioration of established arthritis by gene therapy, such as IL-10 gene therapy.
  • the arthritis may be established arthritis.
  • the present invention also provides use of a cationic liposome to transfect a macrophage in order to deliver an agent of interest.
  • the present invention also provides use of a cationic liposome to transfect a macrophage in order to deliver an agent of interest to a distal site.
  • the present invention further provides use of a cationic liposome to transfect a macrophage in order to deliver an agent of interest to an inflamed site, which may be a distal site.
  • the present invention further provides use of a composition of the present invention which further comprises a macrophage.
  • This composition may be formed in vivo.
  • the present invention also relates to novel compounds and cationic liposomes. According to further aspects of the present invention there are provided a compound according to any one of Figures 10-14; a cationic liposome comprising a mannosylated cationic liposome entity; and a cationic liposome comprising 3-Aza- N -cholesteryloxycarbonylhexane-l,6-diamine (referred to as ACHx), DC-Choi and dioleoylphosphatidylethanolamine (commonly known as "DOPE").
  • ACHx 3-Aza- N -cholesteryloxycarbonylhexane-l,6-diamine
  • DOPE dioleoylphosphatidylethanolamine
  • the present invention also includes pharmaceutical compositions comprising such compounds and cationic liposomes admixed with a pharmaceutically acceptable diluent, carrier or excipient.
  • the present invention further provides the use of such compounds and cationic liposomes for the preparation of a medicament for the treatment of a disorder or condition or disease.
  • Figure 1 shows combined human EL- 10 in situ hybridisation and CDl lb immunocytochemistry. Peritoneal cells taken 4hr after i/p injection of pcD HuIL-10 with liposomes were fractionated into non adherent cells (a) and adherent cells (b) and were hybridised with a probe specific for human EL- 10 D ⁇ A and stained for expression of mouse CD1 lb.
  • Figure 2 shows the structure of DOPE;
  • Figure 3 shows the structure of DC-Choi;
  • Figure 4 is a representation of a macrophage mannosyl receptor;
  • Figures 5 to 9 show mannose containing head groups;
  • Figures 10-14 show mannosylated compounds;
  • Figure 15 shows some polyamines
  • Figure 16 shows spermidine linked to cholesterol via carbamate
  • FIG 17 shows scheme 1 ;
  • Figure 18 shows scheme 2;
  • Figure 19 shows scheme 3;
  • Figure 20 shows enumeration of transfected cells. J774 cells were transfected with pCMV ⁇ and ACHx/DC-Chol:DOPE liposomes and stained 24hr later for ⁇ - galactosidase. Cells were examined under both bright field and phase contrast illumination;
  • Figure 21 shows the effect of IL-10 transfection on LPS stimulated IL-6 production.
  • J774 cells were transfected with pcD murine IL-10 or remained untransfected. After 6hr cells were recovered and added to LPS stimulated J774E cells. 48hr later secreted IL-6 and IL-10 in the culture supernatant were determined by ELISA. Results are mean+SD of one experiment;
  • Figure 22a shows detection of plasmid DNA in mouse tissues by PCR.
  • RNA from tissues of two mice with CIA was taken one day after injection of pcD HuIL-10 with liposomes and analysed by PCR for human IL-10 plasmid.
  • Control was kidney from an untreated mouse;
  • Figure 22b shows digest of human EL-10 PCR product with Bsml.
  • the product of PCR for human IL-10 (using limb RNA) was digested with the enzyme Bsml. Two products of approximate sizes 210 and 140bp are visible;
  • Figure 23 shows detection of human IL-10 cDNA. 5 ⁇ g of RNA taken from the paws of arthritic mice injected 2, 3 or 7 days earlier with pcD HuIL-10 and liposomes and was treated with DNase before cDNA synthesis and PCR analysis for human IL- 10 (upper panel) and actin (lower panel);
  • Figure 24 shows in situ hybridisation of peritoneal exudates. Smears of peritoneal cells taken immediately after injection of plasmid-liposome complexes, or 30min or 4hr later were hybridised with a probe specific for human EL- 10 DNA. As a control cells recovered 4hr after injection of pel vector were treated identically;
  • Figure 25 shows expression of human IL-10 protein in mouse tissues.
  • mice injected i/p with 0.3 mg pcD HuIL-10 and 0.72mg ACHx/DCChol:DOPE liposomes were sacrificed at the times shown and selected tissues analysed by species specific ELISA for the expression of human IL-10 protein. Results are expressed as human IL-10 per gram of tissue. Four mice were analysed on days 1 and 2 and two mice on subsequent days. Background binding of tissue homogenates in the human IL-10 ELISA from four mice injected with pcD vector and ACHx/DCChol:DOPE liposomes and sacrificed on day 1 were determined and subtracted from the IL-10 ELISA values of the pcD HuIL-10 treated mice.
  • Figure 26 shows the course of arthritis after treatment with pcD HuIL-10.
  • mice with CIA of 2 - 4 days duration were injected i/p with 0.3mg of plasmid (either pcD vector or pcD HuIL-10) together with 0.72mg of ACHx/DC-Chol:DOPE liposomes at time zero, or were left untreated.
  • the course of disease was followed by both paw swelling (a) and grade of disease (b);
  • Figure 27 shows the course of arthritis after treatment with pcD HuIL-10 or pel HuIL-10.
  • Mice with CIA of 2 - 4 days duration were injected i/p with 0.3mg of plasmid (either pel vector, pel HuIL-10 or pcD HuIL-10) together with 0.72mg of ACHx/DC-Chol:DOPE liposomes at time zero, or were left untreated.
  • Figure 28 shows sections of PIP joints from arthritic mice, (a) pel vector treated mouse showing inflammatory infiltrates, bone erosion and destruction of the cartilage, (b) Mouse treated with pcD HuIL-10 after the onset of arthritis in this paw shows no evidence of inflammation or erosion and complete protection of the cartilage; 9 Figures 29 to 33 shows N-glycan compounds;
  • Figure 34 is a representation of a glycolipid
  • FIGS 35 and 36 show retrosynthesis schemes
  • Figure 37 shows saccharides a and b;
  • Figure 38 shows saccharides c and e;
  • Figure 39 shows saccharide d
  • Figure 40 shows diethyl squarate coupling
  • Figure 41 shows deprotection conditions
  • Figure 42 shows the preparation of cholesteryl squarate
  • Figure 43 shows the preparation of the oligosaccharide amine
  • Figure 44 shows coupling and deprotection.
  • Figures 45a, b and c show transfection of cells in vitro by mannosylated liposomes.
  • IL-10 synthesis was determined after 48hr by ELISA; IL-10 production by non transfected cells was ⁇ 50pg/ml. Note the different scale for IL-10 production by NIH 3T3 cells.
  • Figure 46 shows PCR for human IL-10 plasmid in tissues of pcD HuIL-10 injected mice. Mice were injected i/p with 0.3mg pcD HuIL-10 and 0.72mg of liposomes. Tissues taken two days later were tested by PCR for the presence of human IL-10 plasmid.
  • ACHx/DCChol:DOPE liposome treated mice Plasmid preparations of limb were used without further dilution, other tissues at the dilutions shown.
  • Figure 47 shows in situ hybridisation for human IL-10 plasmid in spleen.
  • Figure 48 shows increased transfection of spleen tissue using ACHx/DCChol:DOPE liposomes incorporating 5 mol% glycolipid #5.
  • ACHx/DCChol:DOPE liposomes or ACHx/DCChol:DOPE liposomes incorporating 5 mol% glycolipid #5 was analysed by PCR for human IL-10 plasmid.
  • Figures 49a and b show synthesis of human IL-10 after in vitro transfection using
  • ACHx/DCChol:DOPE liposomes incorporating 5 mol% glycolipid #5 (see Fig 14) 3x10 5 adherent cells were transfected with lO ⁇ g pcD HuIL- 10 and 10-40 ⁇ l (12-
  • the cationic liposome may be formed from lipids or other surface active agents which (a) can by themselves form spontaneously into bilayer vesicles in water or (b) can be stably incorporated into the bilayer formed by molecule of type (a).
  • Molecules of type (a) are exemplified by phospholipids.
  • Molecules of type (b) are exemplified by cholesterol and cholesterol derivatives i.e. derivatives which function as cholesterol in a cationic liposome, including derivatives of cholesterol, such as cholesterol sulfate and cholesterol hemisuccinate.
  • cationic liposome forming entities examples include DC-Choi, 1,2-dioleyl-sn- glycero-3-ethylphosphocholine and l,2-dimyristoyl-sn-glycero-3- ethylphosphocholine. Others will be known to those skilled in the art.
  • DC-Choi is a particularly preferred cationic liposome forming entity.
  • the cationic lipids or surface active agents are preferably used in admixture with neutral molecules.
  • a neutral phospholipid useful in the preparation of a cationic liposome is the phospholipid DOPE. Others will be known to those skilled in the art.
  • the cationic liposome forming entity is DC- Chol which is used in a mixture with DOPE.
  • any convenient method for preparing the liposome may be used and are known to those skilled in the art. Examples of such methods include a solvent evaporation process in which a mixture of the liposome-forming compounds are dissolved in a solvent, such as chloroform, which is then evaporated leaving a thin film. The film hydrates in aqueous medium to form liposomes. Sonication may be used to decrease the size of the liposome.
  • a solvent such as chloroform
  • the agent of interest may be loaded into the liposome by a variety of methods which will be known to those skilled in the art.
  • the liposome is prepared using the solvent evaporation process the lipids or surface active agents may be dissolved in the organic solvent, sonicated in the presence of the aqueous phase and the organic solvent removed by evaporation.
  • the agent of interest is a nucleotide sequence it is typically administered in the form of a complex with the cationic liposome.
  • Such complexes are commonly known as cationic liposome-DNA complexes.
  • the complex may be formed by incubating the nucleotide sequence with the cationic liposome.
  • the method of administering the cationic liposomes may be by any liposome adminstration method known in the art. Examples include intravenous, intraperitoneal, intratracheal or intramuscular injection, topical, oral, aerosol or ocular.
  • the cationic liposome may be administered in the form of a pharmaceutical composition.
  • Suitable pharmaceutically acceptable carriers, excipients and diluents are known to those skilled in the art, for example a preferred carrier for intravenous injection is sterile water which may include optional additives such as buffers, preservatives, antioxidants and isotonic salts. 13
  • the agent of interest may be associated with the production of a variety of gene products including protein and RNA.
  • proteins may function as intracellular or extracellular structural elements, hormones, neurotransmitters, growth regulating factors, enzymes, serum proteins, receptors, drugs, immunomodulators, oncogenes, tumor suppressors, toxins, tumor anitgens, antigens, antisense inhibitors, triple strand forming inhibitors or ribozymes etc.
  • the agent of interest is a plasmid containing and capable of functionally expressing a gene which codes for IL-10.
  • the agent of interest is EL- 10.
  • the agent of interest may be a marker for use for example in disease models and assays.
  • the agent of interest may be a drug or pro-drug.
  • agents of interest include cytokines (particularly cytokines with anti- inflammatory roles), inhibitors of cytokine function (natural and synthetic), immune modulators, and inhibitors of transcription factors (particularly of NFKB).
  • the agent of interest is an anti-inflammatory agent.
  • Anti-inflammatory agents include steroids and non-steroids, immunosuppressant agents, methotrexate, azaribine, etretinate, anthralin, cyclosporin and psoralin.
  • gold containing compounds such as aurothioglucose and auranofin.
  • Preferred steroidal anti- 14 mflammato ⁇ es include predmsone, methylpredmsolone, paramethazone, 11- fludrocortisol, t ⁇ amcinolone, betamethasone and dexamethasone
  • Any head group and/or ligand which can increase macrophage uptake of the cationic liposome can be used
  • a screen for such gands may involve compa ⁇ ng macrophage uptake of a catiomc liposome with the prospective ligand and the catiomc liposome without the prospective hgand
  • a comparable screen may be used for head groups Suitable screens use cell staimng techniques or in situ hybridisation techniques Examples of these techniques are desc ⁇ bed m Example 1 below
  • Examples of hgands useful in the present invention include mannose-contammg residues and combinations thereof
  • Examples of head groups useful in the present invention include polyamines and combinations thereof
  • the compound is not synthesised by reacting spermidine and cholesterol chloro formate in CH 2 C1 2 in the presence of N,N-diisopropylethylamine.
  • the cholesterol group can be a cholesterol derivative.
  • cholesterol derivatives include substituted derivatives wherein one or more of the cyclic CH 2 or CH groups and/or one or more of the straight-chain CH 2 or CH groups is/are appropriately substituted. Alternatively, or in addition, one or more of the cyclic groups and/or one or more of the straight-chain groups may be unsaturated.
  • the cationic liposome forming entity is cholesterol. It is believed that cholesterol is advantageous as it stabilises the resultant liposomal bilayer.
  • the cationic liposome forming entity is linked to the head group via a carbamoyl linkage. It is believed that this linkage is advantageous as the resultant liposome has a low or minimal cytotoxicity.
  • the head group is a polyamine group. It is believed that the polyamine group is advantageous because it increases the DNA binding ability and efficiency of gene transfer of the resultant liposome.
  • the polyamine group is a naturally occurring polyamine. It is believed that the polyamine head-group is advantageous because the increased amino functionality increases the overall positive charge of the liposome.
  • polyamines are known to both strongly bind and stabilise DNA. Further, 16 polyamines occur naturally in cells and so it is believed that toxicological problems are minimised.
  • Suitable polyamines include spermidine, spermine, caldopentamine, norspermidine and norspermine. These polyamines are shown in Figure 15.
  • the polyamine is spermidine or spermine as these polyamines are known to interact with single or double stranded DNA.
  • An alternative preferred polyamine is caldopentamine.
  • a preferred compound is spermidine linked to cholesterol via a carbamate linkage.
  • This compound is shown generically in Figure 16. It is believed that the polyamino head group is advantageous for DNA condensation, the carbamate linkage is stable but biodegradable and the cholesteryl group imparts bilayer rigidity.
  • the carbamate linkage may be part of, or an integral component of, the head group.
  • Another preferred compound is spermine linked to cholesterol via a carbamate linkage.
  • the polyamino head group is advantageous for DNA condensation, the carbamate linkage is stable but biodegradable and the cholesteryl group imparts bilayer rigidity.
  • the cationic liposome also comprises a neutral phospholipid - such as DOPE.
  • two or more of the amine groups of the polyamine head groups of the present invention are separated by one or more groups which are 17 not found in nature that separate amine groups of naturally occuring polyamine compounds, such as methylene groups (i.e. preferably the polyamine group of the present invention has un-natural spacing).
  • the free DC-Chol pentamine analogues 23 were prepared by hydrogenolysis of the protecting groups in the usual way (Scheme 3) in 18-42% overall yield.
  • Examples of other compounds capable of forming a cationic liposome include:
  • An especially preferred compound is ACHx. It is surprising that this compound is useful in vivo since in Cooper RG et al ibid it is not disclosed as being among the 23 best when used in vivo.
  • the cationic liposome also comprises DC-Chol and DOPE.
  • a particularly preferred cationic liposome is the novel cationic liposome comprising ACHx, DC- Choi and DOPE (referred to as ACHx/DC-Chol:DOPE).
  • a preferred type of ligand is one which binds to a macrophage mannose receptor ( Figure 4).
  • the ligand comprises mannose, either comprising a single mannose residue (in which case the cationic liposome is referred to as being mannosylated) or the ligand comprises more than one mannose group, for example 4, 5 or 6 mannose residues.
  • Mannosylation has only previously been used in relation to neutral, multilayered liposomes in order to encapsulate drugs (20,21).
  • the present invention now provides mannosylated cationic liposomes.
  • cationic liposomes have the potential to deliver an agent of interest to a site distal from the site of administration of the liposome.
  • cationic liposomes have the potential to deliver an agent of interest to areas of inflammation and that uptake does indeed occur at inflammed sites. Treatments using such cationic liposomes can have a significant beneficial impact on the course of a disease.
  • the mannose residue ligand is linked to cholesterol or a cholesterol group as cationic liposome forming entity.
  • the cholesterol group can be a cholesterol derivative.
  • cholesterol derivatives include substituted derivatives wherein one or more of the cyclic CH 2 or CH groups and/or one or more of the 24 straight-chain CH 2 or CH groups is/are appropriately substituted. Alternatively, or in addition, one or more of the cyclic groups and/or one or more of the straight-chain groups may be unsarurated.
  • the cationic liposome forming entity is cholesterol or even one of the compounds listed above as being capable of forming a cationic lipoosme, such as ACHx.
  • the cholesterol group is linked to the mannose residue via a carbamoyl linkage or a group comprising diethyl squarate.
  • a preferred compound is ⁇ -D-7-(cholesteryloxycarbonyl)amino-3-aza-heptyl-l- rnannopyranoside.
  • the cationic liposome comprises the compound of the present invention and a neutral phospholipid - such DOPE.
  • the liposome also comprises DC- Chol and ACHx.
  • an especially preferred liposome is formed from the cationic liposome of the present invention/ ACHx/DC-Chol:DOPE.
  • a further range of ligands for liposome targeting incorporate structures based on the gp63 major surface glycoprotein of Leishmania mexicana.
  • L. mexicana is an obligate parasite of macrophages and uses at least two cell surface molecules to enter the macrophage, lipophosphoglycan (LPG) and gp63. Both of these structures are formed of complex oligosaccharides consisting of glucosamine, galactose and, principally, mannose (22).
  • the various gp63 structuresof the Leishmania spp have a conserved backbone structure of Manet l-2Man ⁇ l-6Man ⁇ l-4Glc, either unsubstituted or substituted with a single Man residue.
  • LPG has a similar backbone structure (insert), but with a wide variety of side chain subsitutions depending on species and life cycle stage.
  • Antibodies to gp63 inhibit attachment of the infective stage of Leishmania to macrophages (23). 25
  • Preferred ligands include the mannose structures shown in Figures 5-9 .
  • the mannose structures are shown as the corresponding N-glycan in Figures 29-33.
  • the polymannose residue ligand is linked to cholesterol or a cholesterol group cationic liposome forming entity.
  • the cholesterol group can be a cholesterol derivative.
  • cholesterol derivatives include substituted derivatives wherein one or more of the cyclic CH 2 or CH groups and/or one or more of the straight-chain CH 2 or CH groups is/are appropriately substituted. Alternatively, or in addition, one or more of the cyclic groups and/or one or more of the straight-chain groups may be unsaturated.
  • the cationic liposome forming entity is cholesterol.
  • the cholesterol group is linked to the mannose residues via a carbamoyl linkage or a group comprising diethyl squarate.
  • the cationic liposome comprises a compound of the present invention and a neutral phospholipid - such as DOPE.
  • the liposome also comprises DC- Choi and ACHx.
  • an especially preferred liposome comprises the compound of the present invention, and ACHx/DC-Chol:DOPE.
  • Preferably low mole % ratios are used to avoid engagement of the serum mannose binding protein which may 26 inactivate the liposomes.
  • novel compounds of the present invention used in cationic liposome formation are based on a glycolipid.
  • a schematic representation of a glycolipid is shown in Figure 34.
  • pcD which contains a SV40 promoter
  • pcD containing either the murine IL-10 or human IL-10 cDNA
  • Dr Y. Chernajovsky King Institute of Rheumatology, London
  • DNAX Palo Alto, CA.
  • pcD vector To generate control pcD vector, the IL-10 cDNA was excised with Xhol.
  • the second expression plasmid used was pel neo (Promega, Southampton, UK, in which the promoter is from CMV) and a PstI to BamHI full length human IL- 10 cDNA was cloned into pBluescript and subsequently into the EcoRI and Xbal sites of pclneo.
  • the plasmid pCMV ⁇ was obtained from Clontech (Palo Alto, CA).
  • Plasmid DNA was isolated as follows: overnight cultures of bacteria were centrifuged and the pellet resuspended in 20% sucrose, 50mM Tris pH8 with lysozyme (0.8 mg/ml) and incubated on ice for 5 min. EDTA pH8 was added to 60mM and after a further 5 min on ice Tris pH8 was added to 50mM and the suspension incubated for lOmin at 37°C. The resulting spheroblasts were then lysed by alkaline lysis and plasmid was isolated by Qiagen purification according to manufacturers instructions (Qiagen, Dorking, UK). For in vivo transfection an 27 additional caesium chloride gradient purification step was also carried out.
  • plasmid DNA was redissolved in TE with 150mM sodium chloride (TE is lOmM Tris pH8, ImM EDTA pH8) at 2 mg/ml and treated with End-X (Charles River Endosafe Ltd., Margate, UK). Endotoxin contamination of plasmid was determined using the LAL assay (Associates of Cape
  • DCChol :DOPE liposomes were prepared by adding 6 ⁇ mol of 3 ⁇ -[N-(N ',N' - dimethyl amino ethane) carbamoyl] cholesterol (DCChol) and 4 ⁇ mol of dioleoyl L- ⁇ - phosphatidylethanolamine (DOPE) (supplied at lOmg/ml in CHC1 3 by Sigma, Poole, UK) to freshly distilled CH 2 C1 2 (5ml) under nitrogen.
  • DOPE dioleoyl L- ⁇ - phosphatidylethanolamine
  • ACHx/DCChol:DOPE liposomes were formulated from 3-aza-N - cholesteryloxycarbonyl hexane 1,6-diamine (ACHx), DCChol and DOPE.
  • ACHx was synthesised as described previously.
  • ACHx (2.4mg, 4.5 ⁇ mol), DCChol (0.75mg, 1.5 ⁇ mol) and DOPE (0.3ml, 3mg, 4 ⁇ mol; lOmg/ml in CHC1 3 by Sigma) were combined in freshly distilled CH 2 C1 2 (5ml) under nitrogen, before addition of HEPES buffer, sonication and solvent removal, as above. Liposome preparations were stored at 4°C before use. 28 Mannosylated lipid/ACHx/DCChol:DOPE liposomes were formulated as for
  • ACHx/DCChol:DOPE liposomes except that a few mole %, preferably 5 mole %, of the mannosylated lipid were also added before the reverse phase evaporation.
  • Polymannosylated lipid/ACHx/DCChol:DOPE liposomes were formulated as for ACHx/DCChol:DOPE liposomes except that a few mole %, preferably 5 mole %, of the polymannosylated lipid were also added before the reverse phase evaporation.
  • 1774 cells (a mouse macrophage line) were kindly donated by Dr J. Raynes (London School of Hygeine and Tropical Medicine) and available from ATCC and were maintained in RPMI + 5%FCS.
  • the derivative cell line J774E (reference (24) available from P. Stahl, Dept. of Cell Biology and Physiology, University of Washington, St. Louis, MO) was maintained in MEM alpha supplemented with 5% FCS and 60 ⁇ M thioguanine.
  • NIH 3T3 cells (mouse fibroblast cell line, obtained from ATCC) were maintained in RPMI + 5% FCS. For the transfection studies cells were allowed to adhere overnight.
  • LPS Lipopolysaccharide
  • pDNA For in vitro transfections 10 or 15 ⁇ g of pDNA was mixed with the appropriate amount of undiluted liposomes (1.2mg/ml) at the ratios shown, incubated for lOmin at room temperature and added to either adherent cells in RPMI + 5% FCS, or to washed adherent cells in 1ml of Hank s buffered salt solution (HBSS). After lhr at 37°C the HBSS was replaced with RPMI + 5% FCS and the cells incubated for 24hr 29 before harvesting for the in situ /?-galactosidase assay, or 48hr before harvesting of the supernatant for IL-10 ELISA.
  • HBSS Hank s buffered salt solution
  • Human and murine IL-6 and IL-10 were detected by capture assays using pairs of antibodies (Pharmingen, Cambridge Bioscience, Cambridge, UK); # 18071D and 18082D for murine IL-6; 18562D and 18551D for human IL-10 and 18141D and 18152D for murine IL-10 using the manufacturer's protocol. Purified, recombinant IL-6 and IL-10 (Pharmingen) were used as the standards in these assays. Tissue culture supernatants were applied neat and at 1 in 2 dilution. For detection of IL-10 in murine tissues, weighed pieces of post mortem samples were homogenised in RPMI + 5% FCS and the cleared supernatant used in the ELISAs without further dilution.
  • RNA and actin RNA were detected by PCR and RT-PCR respectively. Mice were killed by cervical dislocation and tissue samples snap frozen in liquid nitrogen. For the isolation of total cellular RNA, frozen tissue was thawed directly in TriReagent (Sigma) and samples individually homegenised; RNA was 30 precipitated with isopropanol and redissolved in diethyl pyrocarbonate (depc) treated water. Plasmid DNA (which co-purifies with RNA in this method) was detected by TriReagent (Sigma) and samples individually homegenised; RNA was 30 precipitated with isopropanol and redissolved in diethyl pyrocarbonate (depc) treated water. Plasmid DNA (which co-purifies with RNA in this method) was detected by
  • RNA preparation was further treated with DNase (5units for lOmin at RT; then 15min at 70°C with 2mM EDTA) before reverse transciption of
  • RNA with lOOunits Superscript II (Gibco BRL, Paisley, UK).
  • PCR was performed using 2.5units Taq polymerase (Promega) and 20pmol each of forward and reverse primers of the sequences shown below. 35 PCR cycles were carried out at 95°C for lmin, 58°C for lmin. 72°C for 2min and a final extension of 72°C for 7min. PCR products were analysed on 1% agarose gels. To detect human IL-10 the following primer pair was used: 5' TGAGAACCAAGACCCAGAC 3' and 3' GGGAACTCTTTGGAATAAC 5' yielding a 350bp product.
  • the actin primer pair had the sequences: 5' GTGGGGCGCCCCAGGCACCA 3' and 3'
  • mice were anaesthetised and killed by cervical dislocation. 2ml of HBSS was injected i/p and peritoneal cells aspirated and stored on ice till cells were pelleted by centrifugation. Cells were resuspended, smeared onto microscope slides, air dried and stored frozen.
  • Peritoneal cells were also fractionated into adherent and non adherent populations by culturing in RPMI + 5% FCS on plastic plates (Falcon 3002; Becton Dickinson, UK) for 1 hr, removing non adherent cells by gentle washing in warm HBSS and recovering adherent cells by vigorous washing with ice cold HBSS. Smears of cells were made, air dried and frozen. Tissue for cryostat 31 sectioning was snap frozen, stored at -70°C and warmed to -20°C for sectioning.
  • Post hybridisation washes were of 2xSSC (lxSSC is 0.15M NaCl, 0.015M Na citrate) followed by lxSSC at 45°C and slides were further treated with 0.5% blocking agent (Amersham) in Tris buffered saline (TBS) for 30min.
  • Alkaline phosphatase conjugated anti-fluorescein antibody in 0.5% bovine serum albumin in TBS was applied for 45min and the substrate of nitroblue tetrazolium / 5-bromo-chloro-indolyl phosphate in lOOmM Tris, lOOmM NaCl, 50mMMg 2 Cl pH 9.5 (Amersham) applied O/N at RT.
  • mice Approximately one month after the onset of arthritis mice were killed and paws that had shown evidence of arthritis were removed, formalin fixed and decalcified before sectioning and staining with haematoxylin and eosin. Sagittal sections of the proximal interphalangeal joint were examined for evidence of inflammatory infiltrates and erosion of the bone and cartilage.
  • the efficiency of transfection was assessed by the number of positive cells after transfection with the ?-galactosidase plasmid 33 pCMVb using the in situ ⁇ -galactosidase assay. It was necessary to observe the cells under phase contrast illumination in order to distinguish cells actually producing ⁇ - galactosidase from those that absorbed the X-gal product after fixation.
  • Figure 20 shows that using just lO ⁇ g of pCMVb and 48 ⁇ g ACHx/DC-Chol:DOPE liposomes the majority of cells were transfected and that if the amounts of plasmid and liposomes were increased to 15 ⁇ g and 72 ⁇ g respectively virtually all the cells became /?-galactosidase positive.
  • J774E cells were stimulated with LPS and the amount of murine IL-6 released into the culture supernatant determined by ELISA. Transfection of J774E cells with liposomes resulted in substantial cell death (not shown). Therefore J774 cells (which are less sensitive to the toxic affects of transfection) were transfected with pcD murine IL-10, recovered after 6hr and added to cultures of J774E cells that had been pre-stimulated for 6hr with LPS; control cultures received non-transfected J774 cells.
  • Figure 21 shows that the addition of LPS to co-cultures of J774E and non-transfected J774 cells results in the release of IL-6 and small amounts of IL-10 from the cells.
  • IL-6 IL-6 production was substantially increased (an additional 700pg/ml above endogenous production) and IL-6 production was inhibited by 40% in all the conditions.
  • transfection of cells with pcD murine IL-10 (or pcD HuIL- 10) prior to LPS stimulation resulted in greater inhibitiion of IL-6 release (not shown).
  • mice with active CIA were injected i/p with 0.3mg of pcD Hu IL-10 complexed with 0.72mg 34 of ACHx/DC-Chol:DOPE and sacrificied for tissue sampling at times from 24hr to two weeks post injection.
  • the method used for RNA extraction removed genomic DNA
  • DNA but plasmid DNA co-separated with the RNA and could be detected by PCR.
  • human IL-10 plasmid was present in the kidney, liver, spleen, lung and limbs of mice although not in the draining (inguinal) lymph nodes of the CIA affected limbs nor in the kidney of a non injected animal ( Figure 22a and data not shown).
  • Digest of the PCR product with the enzyme Bsml confirmed that this was indeed human and not murine IL-10 DNA which lacks this restriction site ( Figure 22b).
  • Hu IL-10 plasmid was further detected in the limbs of CIA mice for up to three days after injection although this was of variable amounts (not shown).
  • peritoneal cells taken from an animal that had been injected with pel vector and liposomes were used and these cells did not stain with the human IL-10 specific probe demonstrating the specificity of this technique (Fig 24; top panel).
  • pcD HuIL- 10 and liposomes large amounts of extracellular plasmid was stained and the majority of 35 cells (probably lymphocytes) had a rim of darkly stained plasmid, perhaps by adherence to the cells (Fig 24; second panel). 30min later both extracellular plasmid and the rim stained cells were less apparent but one large cell showed darkly stained cytoplasm (Fig 24; third panel). After 4hr several further changes had occurred.
  • Human IL-10 protein in tissue homogenates was detected by a species specific ELISA and the results confirmed the pattern observed for plasmid detection with high levels of protein expression early on in the kidney (24hr and 48hr), declining to baseline levels by day three, and also in the lung with a raised level of human IL-10 in this tissue remaining at day seven ( Figure 25a). Although plasmid was present in liver and spleen also (not shown), human IL-10 was not seen in these tissues and, interestingly was below the limit of sensitivity of the assay in the serum of mice even at 24hr. However the same samples when tested in an ELISA specific for murine IL-10 (Figure 25b) showed no difference between the tissue samples at different time points, confirming that it was human IL-10 that was detected in these tissues.
  • mice injected with pel vector and liposomes showed a transient drop in disease activity immediately following injection but full disease returned by day six to eight and paw swelling reached a maximum of 1.8mm ( ⁇ 0.4) on day 9 (Fig. 27a).
  • Mice treated with the IL-10 expression plasmid based on pclneo also showed a transient recovery followed by a return to disease, albeit with less paw swelling (maximum of 0.9mm
  • mice injection of the IL-10 expression plasmid used in the first experiment (pcD HuIL- 10) to a third group of CIA affected mice had a substantial affect on the course of disease. Initially, animals again showed an immediate fall in disease activity which in this group was sustained in terms of paw swelling (1.1mm ⁇ 0.2 at day 0 to 0.3mm ⁇ 0.1 at day 9) and, apart from a slight and transient increase at day six to eight, was also observed for the grade of disease (2.7 ⁇ 0.4 at day 0 to 1.2 ⁇ 0.3 at day 9).
  • mice with CIA were treated with liposomes and either pCMV ⁇ or pcD HuIL- 10 plasmids and again the IL-10 plasmid treated mice had less severe disease over a twelve day pe ⁇ od
  • EXPERIMENTAL preparation of a-D-7-(cholestet ⁇ loxycarbonyl)am ⁇ no-3-aza- heptvl-1 -mannopyranoside (referred to as "mannosvlated lipid") 39 1.
  • mannosvlated lipid 3-aza-7-t-butyIdiphenylsilyIoxy-N -cholesteryloxy- carbony v -(phenylmethoxycarbonyl)heptanamine
  • Plasmid DNA (which co- purifies with RNA) was prepared from pieces of tissue of equal size taken two days 45 after injection of plasmid and liposomes and tested by PCR the presence of human
  • IL-10 DNA DNA. PCR was performed on serially diluted preparations to give a semi- quantitative measure of the level of plasmid expression. 2 of 2 mice that received mannosylated lipid/ACHx/DCChol:DOPE liposomes and pcD HuIL- 10 showed a higher level of expression of human IL-10 plasmid in both the spleen and liver compared to 2 mice injected with the ACHx/DCChol:DOPE liposomes and pcD HuIL- 10 (figure 46). These mice did not have active arthritis and we therefore did not anticipate expression in the limbs; however, one mouse did have detectable human IL-10 plasmid in limb tissue also (#4; fig. 46). Expression of human IL-10 plasmid in the kidneys or lungs of mice injected with the two formulations of liposome was not different (fig 46 and not shown).
  • mice are treated 46 with the human IL-10 expression plasmid pcD HuIL- 10 with the mannosylated lipid/ACHx/DC-Chol:DOPE liposomes.
  • disease activity remain lower in the pcD HuIL- 10 treated group throughout the experiment and show an improvement over control aminimals as judged by paw swelling and the grade of arthritis.
  • the compounds are prepared using the following general approach:
  • the appropriate oligosaccharide is prepared using the glycosylation strategy described below in greater detail. After deprotection, the oligosaccharide is converted to the corresponding oligosaccharide amine. The oligosaccharide is then coupled to an appropriately protected cholesteryl amine using diethyl squarate. Any protecting groups may then be removed.
  • Glycosylation strategy the selective activation and glycosylation of saccharides in the presence of other potential glycosyldonors is one of the latest advances in oligosaccharide synthesis.
  • the methodology available for influencing the reactivity of a glycosyl donor is summarized below.
  • Diethyl squarate may be used for the coupling of an oligosaccharide amine to a cholesteryl amine as illustrated in Figure 40.
  • This reaction is described By Kamath VP et al, Glycoconjugate Journal, 1996, 13, 315-319. It is an efficient and reproducible method for the preparation of glycoconjugates even on a very small scale (lmg), uses deprotected saccharides and the reaction can be analyzed by MALDI-TOF mass spectrometry. 48
  • glycolipid formed from (a) is shown in Figure 10.
  • glycolipid formed from (b) is shown in Figure 11.
  • the glycolipid formed from (c) is shown in Figure 12.
  • the glycolipid formed from (d) is shown in Figure 13.
  • glycolipid formed from (e) is shown in Figure 14.
  • J774E cells were stimulated with LPS and the amount of murine IL-6 released into the culture supernatant determined by ELISA. Transfection of J774E cells with liposomes resulted in substantial cell death (not shown). Therefore J774 cells (which are less sensitive to the toxic affects of transfection) were transfected with pcD murine IL-10, recovered after 6hr and added to cultures of J774E cells that had been pre-stimulated for 6hr with LPS; control cultures received non-transfected J774 cells. The addition of LPS to co-cultures of J774E and non-transfected J774 cells results in the release of IL-6 and small amounts of IL-10 from the cells. However, if pcD murine IL-10 transfected J774 cells were present, IL-10 production was substantially increased and IL-6 production was inhibited.
  • ACHx/DC-Chol:DOPE 50 is substituted with any one of the compounds of Figure 10-14.
  • extracted plasmid DNA is detected by PCR.
  • Human IL-10 is present in sites distal to the site of the i/p injection.
  • Digest of the PCR product with the enzyme Bsml confirms that this is human IL-10 DNA.
  • detection of cDNA by PCR confirms that plasmid reaches the limbs of arthritic mice and is transcribed.
  • human IL-10 protein in tissue homogenates is detected by a species specific
  • mice are treated with the human IL-10 expression plasmid pcD HuIL- 10 with any one of the compounds of Figure 10-14/ACHx/DC-Chol:DOPE liposomes.
  • disease activity remain lower in the pcD HuIL- 10 treated group throughout the experiment and show an improvement over control aminimals as judged by paw swelling and the grade of arthritis.
  • IL-10 plasmid rather than mouse in order to trace the distribution of IL-10 plasmid - liposome complexes and the formulation of cationic liposomes (ACHx/DC-ChoEDOPE) that gave the higher transfection efficiency in vitro.
  • plasmid was rapidly distributed to several sites including kidney, liver, spleen, lung and limb (Fig 22 and data not shown) although, interestingly, barely or not at all to the draining lymph nodes of the affected limbs.
  • Previous studies have shown that intravenous and intraperitoneal injections produce reporter gene expression in many tissues including heart, liver, kidney and spleen. Lung tissue was only transfected by i/v administration and lymph nodes only transfected after subcutaneous injection. However, it has not been previously 52 reported that cationic liposomes deliver plasmid to distant sites of inflammation.
  • Monocytes are stimulated to enter the peritoneal cavity after i/p injection of particulate material and the results of the in situ hybridisation staining suggest that predominantly monocytes were transfected by this route of liposome delivery.
  • An influx of monocytes and polymorphonuclear cells was evident after i/p injection (not shown) and it is possible that monocyte uptake was responsible for the rapid clearence of free plasmid-liposome complexes from the peritoneal fluid.
  • IL-10 expression plasmid IL-10 expression plasmid.
  • commercial methods of RNA preparation do not exclude plasmid and it was found necessary to DNase treat plasmid before cDNA synthesis. It is likely that Rogy et al were in fact detecting contaminating plasmid DNA. By careful sample preparation convincing cDNA expression was also demonstrated (Fig 23) in the tissues.
  • IL-10 protein was detected by a species specific ELISA but the high level of endogenous alkaline phosphatase activity in all the tissues examined make this an insensitive method. Probably for this reason we could only detect protein expression in the kidney 24hr and 48hr after injection, although the lung tissue samples had detectable IL-10 at all the time points examined despite cDNA being undetectable. Interestingly IL-10 53 protein was not detected in the serum of these mice at any time suggesting that IL-
  • IL-10 has several modulatory affects on macrophage function including inhibition of pro-inflammatory cytokine synthesis and co-stimulatory activityand has been shown in several studies to have beneficial effects in models of RA.
  • IL-10 plasmid transfection inhibits IL-6 synthesis
  • Animals treated with IL-10 expression plasmid did indeed show an improvement in disease status compared to control treated mice; a moderate amelioration of CIA in the first experiment (Fig 26) but with a significant improvement for up to one month in a second experiment (Fig 27). Initially, it was surprising that mice showed such a rapid decrease in disease activity, with an obvious reduction in swelling after just 24hr.
  • the model of CIA used is an appropiate model of human RA and the treatment protocol that we have used could be applicable to other diseases, conditions or disorders.
  • 55 With reference to Example 2
  • Figure 45 shows that macrophage cell lines in vitro are transfected equally well by the two formulations of liposomes tested and also show that the macrophage lines produce much less transgene product (human IL-10) after transfection than fibroblasts. This is most likely a result of the fibroblasts inherently greater synthetic capacity than a measure of transfection efficiency. However, it also emphasises the surprising in vivo macrophage transfection bu the present invention.
  • Figure 47 shows that the transgene, although given i/p, enters deep into the tissue of at least spleen (and liver, not shown).
  • cationic liposome mediated delivery is a viable method for the delivery of an agent of interest to a distal and/or inflammatory site and the agent of interest is taken up in sufficient quantity to have an effect.
  • 3xl0 5 cells were transfected with lO ⁇ g of pcD Hu IL-10 complexed with the volumes shown (in ⁇ l) of the two formulations of cationic liposomes.
  • the culture supernatant was tested for human IL-10 by species specific ELISA 2 days later and the amounts of IL-10 detected is given in pg/ml.
  • mice tissues were assayed for human IL-10 plasmid, cDNA or protein by PCR, RT-PCR and ELISA respectively. + detected; not detected; na not assayed. 59
  • Table 3
  • Sections of comparable size of spleen and liver taken from mice injected 2 days earlier with either ACHx DCChol:DOPE liposomes or mannosylated lipid/ACHx/DCChol:DOPE liposomes and pcD HuIL- 10 were stained by in situ hybridisation for human IL-10 DNA and/or RNA. The number of foci showing staining are shown; background staining (without specific probe) was nil.
  • the present invention provides novel and targeted liposomes capable of directing an anti-inflammatory gene to macrophages in an inflammatory condition. This is of considerable value to medicine.

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Abstract

L'invention concerne l'utilisation d'une combinaison d'un liposome cationique dérivé et d'un agent intéressant lors de la préparation d'une composition pour introduire l'agent intéressant dans un site distal par rapport au site d'administration de la composition, le liposome cationique dérivé comprenant une entité de formation de liposome cationique et un groupe de tête et/ou un ligand, le groupe de tête et/ou ligand augmentant l'apport macrophage du liposome cationique.
PCT/GB1999/001171 1998-04-17 1999-04-16 Composition comprenant une combinaison de liposomes cationiques derives et d'agent actif Ceased WO1999054344A1 (fr)

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WO2007134819A1 (fr) * 2006-05-18 2007-11-29 Medigene Ag Préparations liposomales cationiques pour le traitement de la polyarthrite rhumatoïde
JP2008505131A (ja) * 2004-07-07 2008-02-21 スタテンス セールム インスティトゥート 糖脂質を用いた、脂質系アジュバント製剤を安定化する組成物及び方法
JP2018532721A (ja) * 2015-09-17 2018-11-08 モデルナティエックス インコーポレイテッドＭｏｄｅｒｎａＴＸ，Ｉｎｃ． 治療剤の細胞内送達のための化合物および組成物
US11066355B2 (en) 2019-09-19 2021-07-20 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11203569B2 (en) 2017-03-15 2021-12-21 Modernatx, Inc. Crystal forms of amino lipids
US11583504B2 (en) 2016-11-08 2023-02-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
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US12077501B2 (en) 2017-06-14 2024-09-03 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
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US12324859B2 (en) 2017-03-15 2025-06-10 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US12396961B2 (en) 2015-12-22 2025-08-26 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents

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JP2008505131A (ja) * 2004-07-07 2008-02-21 スタテンス セールム インスティトゥート 糖脂質を用いた、脂質系アジュバント製剤を安定化する組成物及び方法
WO2007134819A1 (fr) * 2006-05-18 2007-11-29 Medigene Ag Préparations liposomales cationiques pour le traitement de la polyarthrite rhumatoïde
JP2018532721A (ja) * 2015-09-17 2018-11-08 モデルナティエックス インコーポレイテッドＭｏｄｅｒｎａＴＸ，Ｉｎｃ． 治療剤の細胞内送達のための化合物および組成物
US12404232B2 (en) 2015-09-17 2025-09-02 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US11220476B2 (en) 2015-09-17 2022-01-11 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
JP2022008431A (ja) * 2015-09-17 2022-01-13 モデルナティエックス インコーポレイテッド 治療剤の細胞内送達のための化合物および組成物
JP2023164798A (ja) * 2015-09-17 2023-11-14 モデルナティエックス インコーポレイテッド 治療剤の細胞内送達のための化合物および組成物
US12151995B2 (en) 2015-09-17 2024-11-26 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US12396961B2 (en) 2015-12-22 2025-08-26 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US12144895B2 (en) 2016-11-08 2024-11-19 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US11583504B2 (en) 2016-11-08 2023-02-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
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US12077501B2 (en) 2017-06-14 2024-09-03 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US12263248B2 (en) 2018-09-19 2025-04-01 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US12312293B2 (en) 2019-09-19 2025-05-27 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11597698B2 (en) 2019-09-19 2023-03-07 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
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