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WO2002000680A2 - Materiaux et procedes relatifs a l'administration de genes - Google Patents

Materiaux et procedes relatifs a l'administration de genes Download PDF

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Publication number
WO2002000680A2
WO2002000680A2 PCT/GB2001/002859 GB0102859W WO0200680A2 WO 2002000680 A2 WO2002000680 A2 WO 2002000680A2 GB 0102859 W GB0102859 W GB 0102859W WO 0200680 A2 WO0200680 A2 WO 0200680A2
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Prior art keywords
lipid
cationic
gene
liposome
ncc4
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PCT/GB2001/002859
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English (en)
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WO2002000680A3 (fr
Inventor
Kam Man Hui
Hui Gao
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Ncc Technology Pte Ltd.
Stuart, Ian
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Priority to AU2001266189A priority Critical patent/AU2001266189A1/en
Publication of WO2002000680A2 publication Critical patent/WO2002000680A2/fr
Publication of WO2002000680A3 publication Critical patent/WO2002000680A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • 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
    • A61K47/554—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 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to materials and methods involved in gene delivery. Particularly, but not exclusively, the present invention relates to new lipids derived from cholesterol which enhance the efficiency of gene delivery.
  • Gene therapy represents a promising approach for the treatment of inherited or acquired diseases (Refs . 1-4) .
  • one of the most difficult hurdles in achieving effective gene therapy is the requirement for the use of efficient vehicles to deliver the gene of interest into target cells.
  • a diverse spectrum of gene delivery vehicles ranging from replication incompetent viruses to DNA formulated with various delivery vehicles has been utilized (Refs . 5-7).
  • viral vector-based and plasmid DNA-based systems Two approaches have been adopted to introduce exogenous DNA into cells. These are viral vector-based and plasmid DNA-based systems. Each system has its advantages and disadvantages, and all vehicles have been reported to achieve some level of gene delivery. Viral-based vectors have attracted most interests because of their expected high efficiency at mediating gene transfer (Refs. 9,10). Currently, the two most popular viral vectors for gene transfer are replication defective retroviruses and adenoviruses . The retroviral vectors enable high degrees of gene transfer but can transduce only dividing cells (Ref. 11) . Although adenoviral vectors can transfect non-dividing cells, they have been found to induce inflammatory and immune responses and to therefore limit the duration of expression and efficacy of subsequent re-administrations (Refs. 12-14) .
  • non-viral gene delivery systems are based on cationic compounds. These include either cationic polymers or cationic lipids that spontaneously complex with a plasmid DNA construct by means of electrostatic interactions to yield a condensed form of DNA which shows increased stability toward nucleases .
  • cationic compounds include either cationic polymers or cationic lipids that spontaneously complex with a plasmid DNA construct by means of electrostatic interactions to yield a condensed form of DNA which shows increased stability toward nucleases .
  • Several features of the nonviral systems offer many advantages over viral systems, such as the ease of manufacture, safety, stability, lack of vector size limitations, low immunogenicity, and the potential to incorporate targeting ligand (Refs. 15-17).
  • lipids having monocation head groups such as DOTMA (N-[l-(2,3- dioleoyloxy) propyl] -N,N,N-trimethylammonium chloride) , DMRIE (dimyristoyl oxypropyl dimethyl hydroxyethyl ammonium bromide) , DOTAP (1, 2-dioleoyl-3-trimethyl ammonium propane) , and DC-chol (3- ⁇ - [N' ⁇ (N,N' ⁇ t bo o o o o o
  • the present invention provides novel lipids and liposomes which may enhance the delivery into cells of nucleic acid molecules in vi tro and/or in vivo. Further, the invention provides methods for preparing said lipids, and methods for their use in delivery of nucleic acids into cells.
  • the present invention provides a lipid having a steroid hydrophobic domain and a side chain including a heterocyclic ring.
  • the lipid comprises a cholesterol hydrophobic domain, a carbamoyl linker bond and a head group which comprises a heterocyclic moiety and is positively charged and/or is basic such that it can be protonated.
  • the protonated form is generally a salt of an acid.
  • the charged head group is or includes a nitrogen heterocycle.
  • Particularly preferred are the heterocycles morpholine, imidazole, pyridine or piperazine, optionally substituted.
  • a lipid of this aspect of the invention has a formula designated NCC1, NCC3 , NCC4, NCC5, NCC6, NCC9 or NCC10 as shown in Figure 2, or is a derivative and/or a protonated form thereof.
  • Particularly preferred compounds are those designated NCC4 and NCC10, and derivatives and protonated forms thereof.
  • a lipid which comprises a cholesterol hydrophobic domain, a carbamoyl linker bond and a head group which comprises a primary or secondary aliphatic amine or polyamine, optionally protonated to render it positively charged.
  • Particularly preferred lipids of this embodiment have a formula designated NCC2, NCC7 or NCC8 as shown in Figure 2, or are derivatives thereof.
  • a “derivative" of the lipid encompasses a lipid modified by adding or substituting one or more of its side groups, particularly on the steroid domain, without fundamentally altering the essential structure and/or functional activity of the molecule, i.e. without significantly decreasing its efficacy in delivering nucleic acid into cells.
  • the molecule may be modified by substituting a hydrogen atom for a methyl group at one or more positions in the molecule.
  • the degree and pattern of unsaturation of the steroid domain may be varied.
  • Preferred lipids of the present invention include groups, particularly involving nitrogen, which are protonated to a significant extent under conditions of use.
  • the lipids as provided herein may be formulated into liposome preparations.
  • the invention also provides, in a further aspect, a liposome comprising one or more of the lipids described above.
  • the liposomes are preferably prepared by mixing one or more of said lipids with a helper lipid such as DOPE or cholesterol, using standard methods known to those in the art ("Nonviral vectors for gene therapy", Eds. Leaf Huang, Mien-Chie Hung and Ernst Wagner. Academic Press, 1999) .
  • lipids and liposomes of these aspects may be used in methods of nucleic acid delivery, e.g. gene delivery, as described in further detail below.
  • the present invention provides a method of producing a lipid of the first or second aspects, comprising the steps of reacting cholesteryl chloroformate with an amine or a polyamine.
  • the cholesteroyl chloroformate may be dissolved in chloroform in a first step, and an amine or polyamine may then be added in a second step.
  • an amine chloroform solution may be added to a cholesterol chloroformate chloroform solution.
  • the amine or polyamine has a formula corresponding to the lipids shown in Figure 2 (replacing -CO.O-chol with H) , or is a derivative or variant thereof, e.g. having one or more substituents on a heterocyclic ring and/or having less or more unsaturatio .
  • the method includes the further step of purifying the newly synthesised lipid.
  • This is achieved by: recrystallising the lipid with ethanol; recrystallising with ethanol and acetonitrile; recrystallising with methanol then ethanol; or evaporating the solvent under vacuum and dissolving the lipid in ethanol followed by salt formation with HCl gas, filtration and drying.
  • the present invention provides methods of delivering nucleic acid into a cell, comprising the steps of: complexing said nucleic acid with a lipid or liposome as provided herein; and delivering said nucleic acid/cationic lipid complex or nucleic acid/liposome complex into the cell.
  • complexing occurs with lipid in a cationic state, e.g. due to protonation.
  • the nucleic acid may be a DNA or RNA molecule, and may be in the form of an expression vector such that, following delivery, it will be expressed by the cell.
  • the delivery of the nucleic acid is preferably achieved by injection of the complex into the cell .
  • the nucleic acid may be delivered into the cell for a therapeutic, prognostic, diagnostic or prophylactic purpose, e.g. in a method of gene therapy, particularly for treating a cancer or a cardiovascular disease.
  • nucleic acid/lipid complex or nucleic acid/liposome complex may be formulated in a pharmaceutical composition, which may also comprise a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient (i.e. the nucleic acid/lipid or nucleic acid/liposome complex) .
  • the precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes .
  • the present invention provides pharmaceutical compositions comprising one or more of the above lipids or liposomes complexed with a nucleic acid molecule, in combination with a pharmaceutically acceptable excipient.
  • the pharmaceutical composition may be administered to a patient in a method of gene therapy. Administration is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual .
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, and will be clear to one skilled in the art. Examples of the techniques and protocols which are generally applicable here can be found in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. (ed) , 1980.
  • a lipid/nucleic acid or liposome/nucleic acid complex according to the present invention may be used to introduce a particular gene or other nucleic acid molecule into specifically-targetted cells, which will depend on the condition to be treated, and the nucleic acid may contain e.g. regulatory elements which are switched on more or less selectively by these target cells .
  • the present invention provides the use of a lipid as provided herein in the manufacture of a delivery vehicle for delivering a nucleic acid molecule into a cell, which use includes the step of complexing said nucleic acid with a cationic lipid as provided herein.
  • the delivery vehicle thus constructed is, in a preferred embodiment, in the form of a liposome.
  • the liposome is prepared by mixing the cationic lipid with a helper lipid such as DOPE or cholesterol, using standard methods known to those in the art .
  • FIG. 3 Comparison of the efficiencies of transfection of HepG2 cells in vi tro mediated by the cationic lipids NCC1-NCC10. 3 ⁇ g DNA was complexed with the cationic liposomes, which were formulated with the novel cationic lipids synthesized by the present inventors and described herein, and the helper lipid DOPE, at a molar ratio of 6:4 per transfection.
  • FIG. 4 Comparison of the efficiencies of transfection mediated by a variety of non-viral vectors on human HepG2 cells.
  • the cationic liposomes derived from DC-chol, NCC4 and NCC10 contained DOPE at the molar ratio of 6:4. 3mg pCMV-Luciferase DNA was employed per transfection. Cells were harvested 24h following DNA-mediated gene transfer with the various non-viral reagents and the luciferase activities shown are mean values ⁇ SD of triplicates.
  • FIG. 5 Comparison of the efficiencies of transfection on human HepG2 cells following gene delivery with cationic liposomes and adenoviruses .
  • FIG. 6 Comparison of the efficiencies of transfection mediated by DC-chol, NCC4 and NCC10 on various human tumour cell lines.
  • the DNA-liposome complexes in 1ml lactate buffer were added to 5 x 10 5 cells per well in 6-well plates in the absence of serum. After 2h, 20% FBS was added. The cells were harvested 24h after transfection.
  • the luciferase activities shown are mean values ⁇ SD of triplicates .
  • FIG. 7 Effect of FBS on the efficiencies of transfection mediated by cationic liposomes.
  • Cationic liposomes prepared from NCC4, NCC10 or DC-chol at the charge ratios of 0.4, 1.0, 2.63 and 4.0 were employed to transfect pCMV-Luciferase DNA into human HepG2 cells in the presence or absence of FBS .
  • FIG. 8 Effect of mouse serum on the efficiencies of transfection mediated by cationic liposomes.
  • Cationic liposomes prepared from NCC4, NCC10 or DC-chol at the charge ratios of 0.4, 1.0, 2.63 and 4.0 were employed to transfect pCMV-Luciferase DNA into human HepG2 cells in the presence or absence of mouse serum.
  • the mouse serum was obtained from normal BALB/c mice and pooled.
  • NCC4 and NCC10 were less active at high FBS concentrations.
  • Cationic liposomes prepared from NCC4 , NCC10 or DC-chol at the charge ratios of 2.6 and 4.0 were employed to transfect the pCMV-Luciferase DNA into human HepG2 cells in the presence or absence of FBS.
  • lipids for gene delivery. Ten of these lipids, having the structures as shown in Figure 2 were synthesized. They are derivatives of cholesterol .
  • cholesteryl chloroformate was allowed to react either with amines or polyamines to produce the respective cationic lipids ( Figure 1) .
  • HCl salts of NCCl, NCC2 , NCC3 , NCC6, NCC9 and NCC10 were similarly synthesized.
  • Lipids NCC4 and NCC10 were purified by recrystallisation with ethanol and acetonitrile.
  • Lipid NCCl was purified by recrystallisation with methanol first, followed by ethanol, while NCC2 was purified by recrystallisation with ethanol alone.
  • Lipids NCC3 , NCC4, NCC6 and NCC10 were recrystallised with ethanol-acetonitrile, while lipids NCC5, NCC7, NCC8 and NCC9 were purified by evaporating the solvent under vacuum and dissolving the lipid in ethanol, followed by salt formation with HCl gas, filtration and drying.
  • Cationic liposomes were prepared by mixing stocks of the various cationic lipids synthesized above with the helper lipid DOPE (dioleoyl phosphatidylethanolamine) at a molar ratio of 6:4 (cationic lipid:DOPE) under a gentle stream of N 2 , and dried under vacuum overnight as described earlier (Ref. 32) .
  • the dried film of lipid was hydrated in 1ml of 20mM sterile HEPES buffer (pH7.8).
  • HEPES buffer pH 7.8
  • the final volume was diluted to 1ml with the addition of extra HEPES buffer.
  • the liposomes were vortexed for 1 min and hydrated overnight at 4°C. All the cationic liposomes preparations were further sonicated before use as described earlier (Ref. 32).
  • the liposomes/DNA complexes were prepared as earlier described (Ref. 32). Briefly, various amounts of liposomes and DNA were mixed together in 1ml sodium lactated Ringer's buffer (B. Braun Melsungen AG, Melsungen, Germany) and the complexes were allowed to form for a minimum of 15 min at room temperature before being employed for gene delivery.
  • the human cancer cell lines A549, HepG2 , CNE-2, KZ2, and SW837 were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) .
  • the human breast cancer cell line MCF-7 was propagated in RPMI 1640 with 10 % FBS and lO ⁇ g/ml bovine insulin.
  • 1x10 s cells were seeded into each well of a 6-well plate (Nunc, Denmark) . After culturing for 12h, cells were washed twice with PBS. Freshly prepared liposome/DNA complexes were added into each well and incubated at room temperature for 15 min.
  • FBS Hyclone Laboratories, Logan, UT, USA
  • mouse serum obtained from BALB/c mice
  • Adenovirus containing the luciferase gene was obtained from Dr. Matt Cotton (IMP, Vienna, Austria) .
  • the adenoviruses were amplified using human 293 cells.
  • 1 x 10 5 cells were seeded into each well of a 6-well plate 24h before infection and either 10, 100 or 1000 virus particles per cell were then added to each well.
  • Cells were harvested 48h following infection. Luciferase activity and protein assays were performed as described below.
  • the zeta potential (electrokinetic potential) of the newly synthesized cationic liposomes was determined by the ZetaSizer 3000HS (Malvern Instruments, Worcestershire, GB) . Zeta potential correlates with the net surface charge of the liposome complexes, and also reflects the stability of cationic liposomes in solution at various pHs . Calibration was established using the - 50mV DTS50/50 standards from Malvern Instruments as recommended by the manufacturer.
  • the present inventors have employed the cationic lipid DC-chol as a gene delivery vehicle. 32,33 It has been obtained that the gene delivery activity of DC-chol is optimal when used in conjunction with the neutral lipid DOPE at the molar ratio of 6:4 (DC-chol: DOPE) , 19 When the newly synthesized cationic lipids were compared to DC-chol and DOPE at the molar ratio of 6:4 for their ability to deliver the reporter gene pCMV-Luciferase into human HepG2 cells, it was observed that NCC 3, 4, 5, 8 and 10 all gave significantly higher activities than DC- chol (Figure 3) . The increase was even more pronounced for NCC4, NCC5 and NCC10 when compared to DC-chol.
  • NCC4 and NCC10 gave an overall increase of more than 6- and 3- folds respectively in the luciferase activities when compared to DC-chol 24h following gene delivery into HepG2 cells ( Figure 3) .
  • NCC2 and NCC6 demonstrated a reduction in luciferase activity in comparison to DC-chol under the same conditions following gene delivery to HepG2 cells ( Figure 3) .
  • NCC4 and NCC10 were chosen to be further studied since they are relatively easy to be synthesized and demonstrated good level of gene expression following delivery.
  • NCC4 and NCC10 When compared to LIPOFECTAMINE (Gibco-BRL, Gaithersburgh, USA) and the cationic polymer PEI, NCC4 and NCC10 gave more than 2-fold increase in the luciferase gene activity following introduction of the pCMV-luciferase DNA into HepG2 cells ( Figure 4) .
  • adenovirus is one of the most efficient. We have therefore compared the ability 90 4-J
  • NCCIO transfection mediated by NCCIO was enhanced by more than 276 folds in the presence of 1% FBS. It is apparent that NCCIO became more sensitive at higher serum concentrations at the charge ratios of 0.4 and 1.0
  • the anchor residues could be either cholesterol or diacyl chains .
  • the linker within the cationic lipid could be in the form of a urea, amine, amide, ether or ester bond. 22 The linker bond has been found to have some correlation with the stability of the cationic liposomes. It has been reported that when a carbamoyl bond is employed as the linker bond, the lipids derived are degradable and therefore would be potentially less toxic to the target cells both in vi tro and in vivo . 19
  • the positively charged head group of a cationic lipid appears to be the most important domain in determining the overall efficiency of gene delivery characteristics for the particular cationic lipid.
  • Lipids bearing linear amines or polyamines as positively charged head group exhibit good gene delivery activities. 23 This is especially true for cationic lipids that demonstrate an overall T-shape configuration. 23 Therefore, when the orientation of the amine or polyamine head group is structurally perpendicular in relation to the lipid anchor, the efficiency of the lipid to mediate DNA gene delivery will be enhanced.
  • cationic lipids containing linear amine (NCC2) or polyamine (NCC7 and 8) as head group were also synthesized and compared to lipids having heterocyclic head groups for their ability to act as gene delivery vehicles (Figure 2) .
  • Cationic liposomes prepared from the cationic lipids NCCl, NCC3 , NCC4, NCC5 and NCC10 that contain heterocycles as head groups gave better or similar efficiency of gene transfer in comparison to DC- chol.
  • NCC6 which gave a poorer efficiency in comparison to DC-chol ( Figure 2) .
  • cationic lipids with linear primary amines or polyamines as the head group were less active than lipids having heterocycles as the head group in their structure with reference to their ability to deliver DNA into target cells.
  • lipids with piperazine (NCCl, 5, 9 and 10) and morpholine (NCC4) are relatively more active ( Figure 3) .
  • NCC4 and NCC10 are the most active of the ten newly synthesized cationic lipids.
  • NCC9 has a piperazine group as the head group, it is not efficient in gene delivery.
  • NCC9 contains two cholesteryl groups and as a result, it is possible that its overall structure might be too bulky to interact with DNA (Figure 2) .
  • cationic lipids with pyridine as their head group for example NCC6, are less active.
  • the positively charged head group was generally believed to allow interactions between the cationic lipid and the negatively charged DNA, and also the cell membrane through charge/charge interactions.
  • the presence of nitrogen and oxygen atoms in the heterocyclic ring might further contribute to this charge/charge interaction of liposome and plasmid DNA and stabilize the binding between the cationic liposomes and DNA.
  • NCC4 and NCC10 were the most efficient gene delivery vehicles. This was also true when they were employed to transfect cell lines such as HepG2 (human liver cancer cell line) and KZ2 (human melanoma cell line) that are generally very difficult to transfect with other reagents including DC-chol, PEI, and LIPOFECTAMINE ( Figure 6) .
  • NCC5 When first studied, the cationic lipid NCC5 gave high efficiency of transfection with HepG2. However, its activity decreased very sharply on storage. A likely explanation is that NCC5 not stable in such a formulation.
  • NCC4 and NCC10 can mediate efficient gene delivery in vi tro and mediate efficient gene delivery followed intra-splenic injection in vivo.
  • both NCC4 and NCC10 could withstand serum inactivation in vi tro by changing the DNA/lipid charge ratios.
  • both NCC4 and NCC10 demonstrate lower levels of gene expression following tail vein injection (data not shown) .
  • the conditions employed for intra-splenic injection could not be directly employed for systemic gene delivery and the conditions established in vi tro to demonstrate serum sensitivity ( Figures 7 and 8) could not be applied directly in vivo .
  • NCC4 cationic liposome
  • mice (4 to 6 weeks old) were cared for and used in accordance with institutional guidelines. Mice were housed for 1 week before treatment . Healthy mice were injected with NCC4 or Ringer's buffer (B. Braun Melsungen AG, Germany) under aseptic conditions.
  • the present invention provides a method of designing functional analogues of the cationic lipids as provided herein, which are effective in delivering nucleic acid molecules into cells, said method comprising:
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.
  • a range of sources e.g. spectroscopic techniques, X-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modelling process.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the compound is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the functional activity of the lead compound.
  • the compound or compounds found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it . Further optimisation or modification can then be carried out to arrive at one or more final compounds for further testing or optimisation, e.g. in vivo or clinical testing.
  • Miller DG Adam MA
  • Miller AD Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 1990;
  • CFTR gene to lung of nonhuman primates toxicity study. Hum Gene Ther 1996; 4: 771-780.

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Abstract

Nouveaux lipides pour former des liposomes destinés à être utilisés comme véhicules pour administrer des acides nucléiques, p.ex. en thérapie génique; ils possèdent une structure avec une domaine stéroïde (cholestéryle) lié à travers une liaison carbamoyle à un groupe de tête qui est positivement chargé pendant l'utilisation (en règle générale, par protonation). Le groupe de tête comprend de préférence un hétérocycle d'azote, p.ex. un système de noyau pipérazine ou morpholine.
PCT/GB2001/002859 2000-06-26 2001-06-26 Materiaux et procedes relatifs a l'administration de genes WO2002000680A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
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WO2002066490A3 (fr) * 2001-02-21 2003-04-24 Novosom Ag Derives de sterols cationiques, liposomes sensibles au ph et contenant ces derives et procede de permettant de charger les liposomes de principes actifs
CN115487168A (zh) * 2022-09-27 2022-12-20 浙江大学 一种基于含氮杂环胆固醇衍生物的脂质纳米颗粒及其应用
CN117003808A (zh) * 2022-04-28 2023-11-07 北京科兴中维生物技术有限公司 一种阳离子脂质化合物及其制备方法和用途
WO2025140596A1 (fr) * 2023-12-29 2025-07-03 康希诺(上海)生物研发有限公司 Composé lipidique cationique ionisable pour l'administration et la composition d'acides nucléiques et son utilisation
WO2025140618A1 (fr) * 2023-12-29 2025-07-03 康希诺(上海)生物研发有限公司 Nanoparticule lipidique pour l'administration d'acide nucléique, son procédé de préparation et son utilisation

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6071890A (en) * 1994-12-09 2000-06-06 Genzyme Corporation Organ-specific targeting of cationic amphiphile/DNA complexes for gene therapy
JPH11507352A (ja) * 1995-06-07 1999-06-29 ジンタ・インコーポレイテッド 新規カルバメート基本カチオン性脂質
WO1997004748A2 (fr) * 1995-08-01 1997-02-13 Advanced Therapies, Inc. Enveloppes virales artificielles renforcees pour l'apport de substances therapeutiques dans les cellules
DE69708868T2 (de) * 1996-04-12 2002-07-18 University Of Pittsburgh, Pittsburgh Neue kationische cholesterin derivate mit zyklischen polaren gruppen
US5912239A (en) * 1997-04-04 1999-06-15 Genzyme Corporation Imidazole-containing cationic amphiphiles for intracellular delivery of therapeutic molecules

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002066490A3 (fr) * 2001-02-21 2003-04-24 Novosom Ag Derives de sterols cationiques, liposomes sensibles au ph et contenant ces derives et procede de permettant de charger les liposomes de principes actifs
US7312206B2 (en) 2001-02-21 2007-12-25 Novosom Ag Sterol derivatives, liposomes comprising sterol derivatives and method for loading liposomes with active substances
CN117003808A (zh) * 2022-04-28 2023-11-07 北京科兴中维生物技术有限公司 一种阳离子脂质化合物及其制备方法和用途
CN115487168A (zh) * 2022-09-27 2022-12-20 浙江大学 一种基于含氮杂环胆固醇衍生物的脂质纳米颗粒及其应用
WO2025140596A1 (fr) * 2023-12-29 2025-07-03 康希诺(上海)生物研发有限公司 Composé lipidique cationique ionisable pour l'administration et la composition d'acides nucléiques et son utilisation
WO2025140618A1 (fr) * 2023-12-29 2025-07-03 康希诺(上海)生物研发有限公司 Nanoparticule lipidique pour l'administration d'acide nucléique, son procédé de préparation et son utilisation

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