WO2000012454A9 - Nouveaux lipides cationiques - Google Patents

Nouveaux lipides cationiques

Info

Publication number
WO2000012454A9
WO2000012454A9 PCT/US1999/019629 US9919629W WO0012454A9 WO 2000012454 A9 WO2000012454 A9 WO 2000012454A9 US 9919629 W US9919629 W US 9919629W WO 0012454 A9 WO0012454 A9 WO 0012454A9
Authority
WO
WIPO (PCT)
Prior art keywords
compound
cells
lipids
composition
transfection
Prior art date
Application number
PCT/US1999/019629
Other languages
English (en)
Other versions
WO2000012454A1 (fr
Inventor
Alberto Haces
Original Assignee
Alberto Haces
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Alberto Haces filed Critical Alberto Haces
Priority to AU55881/99A priority Critical patent/AU5588199A/en
Publication of WO2000012454A1 publication Critical patent/WO2000012454A1/fr
Publication of WO2000012454A9 publication Critical patent/WO2000012454A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/08—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/12—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/10—Phosphatides, e.g. lecithin

Definitions

  • This invention relates to polycationic lipids useful for the delivery (transfection) of nucleic acids (DNA,RNA) and other negatively charged or neutral molecules into living cells, either in vivo or in vitro.
  • Liposome aggregates made -with polycationic lipids are useful structures capable of complexing with negatively charged macromolecules such as DNA or RNA. These complexes can be taken up by living cells and then, once inside the cytosol, through an unknown mechanism, they are presumed to migrate into the cell nucleus. In the nucleus, there are enzymes capable of "reading” and “expressing” the message coded by the nucleic acids so delivered and produce new proteins, which were not being produced by the cell before the transfection of the foreign nucleic acid. When cells so transfected divide and their daughter cells still have the capability to produce the proteins encoded by the initially transfected DNA, the transfection is said to be stable.
  • the new DNA has stably integrated into the cell nucleus changing the cell's genetic make-up.
  • the parent cells can produce the protein encoded by the transfected DNA, but their daughter cells are not capable of expressing that tranfected DNA, the transfection is said to be transient.
  • RNA transfection is always transient. Stable transfection of human or animal cells is the basis of so called gene therapy, since cells which are deficient in a crucial protein for the organism's survival could in principle be repaired by stably transfecting the DNA needed to produce the absent protein.
  • Another type of potential use of DNA/RNA transfection for therapy is antisense therapy.
  • nucleic acid capable of adhering (hybridizing) to defective DNA (or RNA) which is being expressed by the cells to produce an undesired protein, such as an oncoprotein (cancer causing protein)
  • an undesired protein such as an oncoprotein (cancer causing protein)
  • This method of therapy does not change the genetic make-up of the cell, but blocks the effect of the genetic disorder already present in the cell's genome.
  • polycationic lipids for use in human therapy, there is already a well established market for these types of chemicals in the research products field. They are currently being used by researchers to deliver nucleic acids and proteins into cells in order to study how the expression of different genes affect cell growth and function.
  • ex vivo and in vivo transfection There are two possible ways to deliver DNA into cells for gene therapy : ex vivo and in vivo transfection.
  • ex vivo modality cells from a patient are removed from the body, cultured and transfected in vitro. Then, the cells are returned into the patient where the beneficial DNA message is hopefully expressed.
  • the DNA is delivered directly into the patient, which makes this procedure simpler and less expensive.
  • the only effective way to deliver DNA in vivo is by using a virus which naturally infects cells of a specific organ (targets that organ) within the body, and whose genetic make up has been modified by adding the DNA beneficial to the patient.
  • the virus can incorporate the new DNA in the genome of the cell (stable transfection) and the parent cell and its daughters can express the beneficial protein.
  • the pathological component of the virus has been deleted before the patient is exposed to such a virus and only the targeting component left intact. Virus can do this process sometimes with nearly 100% efficiency.
  • there are risks associated with their use since they can produce immunological reactions which may be fatal to the patient; the DNA incorporation in the cell's genome is random, therefore it might disrupt needed genes or activate oncogenes; they are also difficult to mass produce, and so forth.
  • Liposomes or lipid aggregates do not have the side effects of viruses, but are not as efficient as viruses are. There is a constant need to develop newer lipids that can approach the efficiency of viruses without their undesirable side effects (E. Marshall, Science 269,1050 (1995)) . There are several lipids for nucleic acids transfection already in the market. The most relevant of these lipids are: (a) DOTMA (N-[l-(2,3-dioleoyloxy)propyl]- N,N,N - trimethylammonium chloride, U.S. Pat. No. 4,897,355 to D.
  • DOTMA N-[l-(2,3-dioleoyloxy)propyl]- N,N,N - trimethylammonium chloride
  • DOGS is sold under the trade name TransfectamTM by the Promega Corp. Madison, WI );
  • DOSPA (2,3-dioleyloxy-N-[2(sper- ⁇ necarboxyamido)ethyl]-N,N ⁇ iimethyl-l-prop- aminium trifluoroacetate, U.S. Pat. No. 5,334,761 to Gebeyehu, G. et al.);
  • DDAB Dimethyloctadecylammoniumbromide, U.S. Pat. No.
  • DOGS dioleolylphosphatidylethanolamine
  • DOPE dioleolylphosphatidylethanolamine
  • lipids are useful as transfection reagents for nucleic acids, oligonucleotides, mononucleotides, polypeptides, and proteins.
  • some of these lipids are also useful as more effective detergents for cleaning and as vehicles in the cosmetic field.
  • novel oxo and sulfinyl backbone substituted polycationic lipids with ammonium, guanidinium and amidinium positively charged moieties as anchoring groups having Formula I as shown below and in WO 97/42819 (International Application No. PCT/US97/09093), the contents of which is fully incorporated herein by reference.
  • the present invention also encompasses a series of novel phosphatidyl and glyceryl guanidinium cationic lipids having the formula 2 shown below and in Figure 8.
  • the backbone of the substituted polycationic lipis is comprised of a polyether in accordance with Formula 3 as shown in Figure 9:
  • Figure 1 is a graphic representation of Scheme I for the preparation of polycationic lipids of the present invention.
  • Figure 2 is a graphic representation of Scheme II for the preparation of polycationic lipids of the present invention.
  • Figure 3 is a graphic representation of Scheme HI for the preparation of polycationic lipids of the present invention.
  • Figure 4 is a graphic representation of Scheme IN for the preparation of polycationic lipids of the present invention.
  • Figure 5 is a graphic representation of Scheme V for the preparation of polycationic lipids of the present invention.
  • Figure 6 is a graphic representation of Scheme VI for the preparation of polycationic lipids of the present invention.
  • Figure 7 is a graphic representation of Formula 1, which represents a series of polycationic lipids of the present invention.
  • Figure 8 is a graphic representation of Formula 2, which represents a second series of polycationic lipids of the present invention.
  • Figure 9 is a graphic representation of Formula 3, which represents a third series of polycationic lipids of the present invention.
  • Figure 10 is a graphic representation of Formula 4, which represents a fourth series of polycationic lipids of the present invention.
  • Figure 11 is a graphic representation of compound 13a of the present invention.
  • Figure 12 is a graphic representation of compound 13d of the present invention.
  • Figure 13 is a graphic representation of compound 5a of the present invention.
  • Figure 14 is a graphic representation of compound 13b of the present invention.
  • Figure 15 is a graphic representation of compound 6d of the present invention.
  • Figure 16 is a graphic representation of compound 15 of the present invention.
  • the compounds of the present invention can be used alone or in mixtures with other compounds
  • liposome forming compounds (co-lipids) to prepare lipid aggregates which are useful to deliver
  • Colipids are compounds capable of producing stable liposomes
  • colipids are preferably neutral, although they can alternatively be positively or negatively charged.
  • Such examples include phospholipid-related materials, such as
  • lecithin as lecithin, phosphatid lethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphinogomyelin, cephalin, cardiolipin, phosphatidic
  • POPE dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)cyclohexane-l- carboxylate
  • DOPE-mal dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)cyclohexane-l- carboxylate
  • Additional non-phosphorous containing lipids are, e.g.,
  • the lipids depicted in Formula I have a hydrocarbon backbone substituted with
  • heteroatoms which are sterically smaller, but equally or more flexible as the methylene group that they replace. This feature makes these new lipids fit more closely to the macromolecule to be delivered to the cells. This closer fit combined with the polycationic nature of the
  • heteroatoms are hydrophilic; thus,
  • hydrophilic backbone being linear allows for proper
  • one of the preferred embodiments is compound 5a, as shown in
  • FIG 13 which has four hydrophobic tails and two positive charges (two tails per charge).
  • Figures 11 and 14 have fewer hydrophobic tails and a more hydrophobic backbone (no heteroatom substitution, but less hydrophobic overall). However, these compounds have moieties which mimic small natural biological effectors such as the neurotransmitters gamma
  • GAB A amino butyric acid
  • acetylcholine etc.
  • embodiments are compound 13a and compound 13d, as shown in Figures 11 and 12.
  • guanidinium and amidinium moieties have the ability to form hydrogen bridges with the nucleic acids' bases (guanidinium salts are used as
  • the guanidino moiety can also be used to target neural cells, since compounds such as Guanethidine, which possess such a functional group,
  • guanidinium or amidinium functionality is used as the positively charged anchoring group, and they also lack a quaternary ammonium group at the C-1 position of the glycerol
  • DOPE DOPC
  • liposomes with other cationic lipid compounds liposomes with other cationic lipid compounds.
  • LAH lithium aluminum hydride
  • amidine(6e) the dinitrile (4e) with anhydrous hydrogen chloride in ethanol, followed by treatment of the imidoester so obtained with ammonium hydroxide.
  • this guanidinium derivative can be alkylated with, for example, iodomethane to produce the corresponding quaternary ammonium salt.
  • Guanidinium compound (10) can be
  • N,N,N',N'-dioctadecyl-2,2'-oxy bis ethylamine (19mg, 0.017mmol) was dissolved in iodomethane (1ml) inside a capped thick-wall test tube, and the resulting solution heated for
  • Example 6 Synthesis of N.N,N,N',N'.N'-cyanoethyloctadecylmethyl-2,2'-oxybis ethyl ammonium iodide (5e).
  • Example 7 Synthesis of N,N.N,N'.N'.N'- acetoxyethyldioctadecyl-2.2'-oxybis ethyl ammonium iodide(5f).
  • 1,4-diamino butane (2g, 22 mmol) was cooled to 0C (ice bath) and to this solid was
  • Example 15 Synthesis of N,N'.N".N'"-tetrapalmyltetraacetoxyethylspermine iodide salt i3a): A solution of tetrapalmylspermine (Haces et al.,PCT Int. Pub. No WO/95/ 17373),
  • N,N' ' ' -4-bromobutyryl-N,N' ,N" ,N' ' '-tetrapalmylspermine (350mg,0.25 mmol) was dissolved in iodomethane (3ml) and the resulting solution let react for 2 days at room
  • Lipids were formulated by mixing appropriate molar amounts of the active lipid
  • DOPE ethanolamine
  • Plasmid pCMV a-gal which contains the
  • E. Coli a-galactosidase (gene) under the control of the powerful cytomegalovirus promoter was purchased from Clontech, Inc.
  • Primary cells were from human tracheal isolates and neonatal foreskin.
  • Example 20 Transfection of HepG2 and HeLa cells.
  • lipids aliquats (1,3 and 5 ⁇ l of 1 mg/ml liposome in water) were diluted in polystyrene tubes containing lOO ⁇ l of serum-free,
  • the cells for a final volume of 1 ml and a final concentration of 10% FCS, pen/strep, 50 ⁇ g/ml
  • Keratinocvtes Cells were grown in serum free medium (SFM) and plated on 35 mm plates (6 wells) such that the confluence after 24h was above 50%. Plasmid reporter (2 ⁇ g and 5 ⁇ g, respectively) was mixed with variable amounts of liposomes (see tables TV and V) and the
  • the cells were transfected during 4h and 6h, respectively.
  • the DNA/liposome complex was removed by rinsing with SFM and the cells incubated for 48h under normal growth conditions and then assayed for the appropiate marker.
  • Example 22 Transfection and CAT assay of Turkat Cells (suspension cells).
  • the cell suspension culture was transferred to a 50 ml conical tube and centrifuged at 400g for 10 min. The cells were washed twice by aspirating off the supernatant and gently
  • Example 23 Assay for transient transfection (adherent cells).
  • the cells were washed twice with Dulbecco's PBS and stained with freshly prepared
  • the cells were stained with 0.5 ml of ⁇ -galactosidase histochemical stain (0.1% x-gal, 5mM potassium ferrocyanide, 5mM potassium ferrocyanide, 2mM MgCl 2 in PBS)
  • TMTPS Compound 3 in PCT Int. Pub. No. W095/ 17373. Haces, A. et al.) in HepG2 (human hepatocarcinoma) and HeLaS3 (human cervical carcinoma) cells, respectively, and
  • Primary cells are cells that are freshly isolated from humans or animals and which, unlike the cultured cell lines, reflect the potential behavior of a compound in vivo
  • tracheobronchial cells are involved in the genetic disease cystic fibrosis.
  • Table V depicts the percentages of ⁇ -gal positive cells (absolute number) which
  • keratinocytes are also a potential target cells for genetic therapy (Fenjves, E.S. et al., Hum
  • Cells were plated in 48 well plates at a density of 1 x 10 per well in 0.5 ml of growth medium. After 24h, the cells were washed with serum free medium and transfected with a suboptimal amount (150 ng) of plasmid pCMV- ⁇ gal. using 1,3 and 5 ⁇ l (1,3 and 5 ⁇ g) of lipid formulation. The amount giving the highest level of transfection efficiency is shown. The experiment was run in triplicate.
  • Cells were plated in 48 well plates at a density of 1 x 10 5 per well in 0.5 ml of growth medium. After 24h, the cells were washed with serum free medium and transfected with a suboptimal amount (150 ng) of plasmid pCMV- ⁇ gal. using 1,3 and 5 ⁇ l (1,3 and 5 ⁇ g) of lipid formulation. The amount giving the highest level of transfection efficiency is shown. The experiment was run in triplicate.
  • Lipid/DOPE molar ratio
  • Optimal Liposome Amount CAT Activity (mU/well) _g)
  • 35 mm plates were inoculated with cell isolates and transfected at 90% confluence. 2 ⁇ g of firefly luciferase plasmid were mixed with 12 ⁇ g of the lipids and then added to the cells. After 5h, the transfection was quenched by removal of the DNA/Lipid complex and the cells incubated for 72h. Cells were lysated and aliquats assayed for luciferase activity. Cell toxicity, determined by the trypan blue method, was below 5% for lipids 6d and 13d and between 10-20% for Lipofectin.
  • Lipid/DOPE molar ratio
  • Cells were seeded at 2xl0 5 /well in 35 mm wells and transfected the next day. 5 ⁇ g of ⁇ gal DNA were mixed with the appropiate amount of lipids and added to the cells. After 4h, the medium was replaced and the cells incubated for additional 48h and then assayed. Blue cells were observed under the microscope and counted.
  • lipids have a polyether backbone which generally
  • polyether functionality which is also present in
  • polyethylene glycol PEG
  • PEG polyethylene glycol
  • N,N -dipalmityl-triethyleneglycoldiamine (240mg, 0.4mmol) was dissolved in an
  • DOPE colipid dioleoylphosphatidylethanolamine
  • Lipid/DOPE (molar ratio) Liposome Amount ( ⁇ g) ⁇ -Gal Positive Cells (%)
  • Cells were plated in 35 mm well plates at a density of 1 x 10 per well in 1 ml of growth medium. After 24 h (ca 70% confluence), the cells were washed with serum free medium and transfected with 2 ⁇ of plasmid pCMV- ⁇ gal. Cells were assayed 48 h after transfection for the ⁇ gal gene protein.
  • Lipid/DOPE (molar ratio) DNA/Liposomes ⁇ Gal Positive Cells (%)
  • Cells were transfected with the plasmid pCMV- ⁇ gal in 35 mm well plates at 80% confluence in 1 ml of growth medium. After 24 h, the cells were assayed for the expression of the ⁇ gal gene protein.
  • COS1 cells and CHO cells following essentially the same procedures described in connection with Examples 20-22 using the amounts of liposomes and slight changes to the procedure as shown in connection with Tables VI and VII respectively.
  • CHO cells were transfected employing compound 24 as a transfecting agent.
  • lipids having a glyceryl backbone are provided.
  • These glyceryl backbone based lipids have a dicationic bicyclic triethylene diammonium polar head.
  • the high charge density makes the binding of this class of lipids
  • DOTMA Lipofectin reagentTM, Life Technologies, Inc.
  • Scheme V of Figure 5 shows the general synthesis of glyceryl backbone
  • This material was formulated with a colipid dioleoyl phosphatidyl ethanolamine (DOPE) at a 1:1.5., Lipid:DOPE, respectively.
  • DOPE colipid dioleoyl phosphatidyl ethanolamine
  • compound 26 is produced by acylating the starting diol with palmitoyl chloride in the presence of triethylamine to
  • acyl halides e.g. methyl iodide or activated esters (e.g. tosylates or triflates) instead of the acyl halides
  • Liposomes containing lipids produced in accordance with the present invention Liposomes containing lipids produced in accordance with the present invention and
  • Table VIII shows remarkable transfection efficiencies for liposomes derived from
  • compound (6d) has proven to be an outstanding reagent for the transfection of transfection-refractory or difficult-to-transfect cells types
  • liposome reagents of the previous art were considered superior if transfection efficiencies

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Medicinal Preparation (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

La présente invention concerne des lipides cationiques utilisés pour transfecter des macromolécules, ces lipides présentant un polyéther ou un squelette glycérique. En outre, ces lipides peuvent être contenus dans un liposome pour transfecter efficacement des types variés de cellules et améliorer l'efficacité de la transfection. Par ailleurs, cette invention concerne des compositions renfermant lesdits lipides ainsi que leurs procédés d'utilisation.
PCT/US1999/019629 1998-08-27 1999-08-27 Nouveaux lipides cationiques WO2000012454A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU55881/99A AU5588199A (en) 1998-08-27 1999-08-27 Novel polycationic lipids

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US9807398P 1998-08-27 1998-08-27
US60/098,073 1998-08-27

Publications (2)

Publication Number Publication Date
WO2000012454A1 WO2000012454A1 (fr) 2000-03-09
WO2000012454A9 true WO2000012454A9 (fr) 2000-06-15

Family

ID=22266831

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/019629 WO2000012454A1 (fr) 1998-08-27 1999-08-27 Nouveaux lipides cationiques

Country Status (2)

Country Link
AU (1) AU5588199A (fr)
WO (1) WO2000012454A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9358300B2 (en) 1998-11-12 2016-06-07 Life Technologies Corporation Transfection reagents

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7664119B2 (en) * 2001-03-30 2010-02-16 Intel Corporation Method and apparatus to perform network routing
US7923207B2 (en) 2004-11-22 2011-04-12 Dharmacon, Inc. Apparatus and system having dry gene silencing pools
CN103739510B (zh) * 2014-01-16 2015-11-18 中国科学院化学研究所 含氨基末端及双胺基的表面活性剂的制备方法及分离方法
CN106572974B (zh) 2014-07-15 2021-04-23 生命技术公司 用于将分子有效递送到细胞的具有脂质聚集体的组合物和方法
JP2018035121A (ja) * 2016-09-02 2018-03-08 東ソー株式会社 カチオン性ジェミニ型界面活性剤
WO2019176079A1 (fr) * 2018-03-16 2019-09-19 株式会社 東芝 Composé biodégradable, particule lipidique, composition contenant des particules lipidiques et kit
CN110256287B (zh) * 2019-05-08 2023-03-10 山东汉鸿新材料科技有限公司 高含量n-脂肪烷基-1，3-丙撑二胺的合成方法
US10501404B1 (en) 2019-07-30 2019-12-10 Factor Bioscience Inc. Cationic lipids and transfection methods

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4897355A (en) * 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
FR2645866B1 (fr) * 1989-04-17 1991-07-05 Centre Nat Rech Scient Nouvelles lipopolyamines, leur preparation et leur emploi
US5334761A (en) * 1992-08-28 1994-08-02 Life Technologies, Inc. Cationic lipids

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9358300B2 (en) 1998-11-12 2016-06-07 Life Technologies Corporation Transfection reagents

Also Published As

Publication number Publication date
AU5588199A (en) 2000-03-21
WO2000012454A1 (fr) 2000-03-09

Similar Documents

Publication Publication Date Title
US6399663B1 (en) Highly packed polycationic ammonium, sulfonium and phosphonium lipids
Niculescu-Duvaz et al. Structure-activity relationship in cationic lipid mediated gene transfection
US6656498B1 (en) Cationic liposomes for gene transfer
JP4340330B2 (ja) トランスフェクション剤としてのポリカチオン性ステロール誘導体
US6638529B2 (en) Amide-based cationic lipids
AU731503B2 (en) Cationic amphiphile compositions for intracellular delivery of therapeutic molecules
AU6649496A (en) Novel amide-based cationic lipids
WO1996018372A2 (fr) Amphiphiles cationiques et plasmides destines a la liberation intracellulaire de molecules therapeutiques
EP1328254A2 (fr) Formulations de lipides pour une administration ciblee
HUT77171A (hu) Lipopoliaminok mint transzfekciós ágensek, és ezeket tartalmazó gyógyászati készítmények
US6281371B1 (en) Lipopolyamines, and the preparation and use thereof
WO1997042819A9 (fr) Lipides cationiques destines a la transfection de molecules neutres ou chargees negativement a l'interieur de cellules vivantes
WO1997042819A1 (fr) Lipides cationiques destines a la transfection de molecules neutres ou chargees negativement a l'interieur de cellules vivantes
WO2000012454A9 (fr) Nouveaux lipides cationiques
AU8113398A (en) Novel lipid complexes for transferring at least one therapeutically active substance, in particular a polynucleotide, into a target cell, and use in gene therapy
WO1998050418A1 (fr) Amphiphiles cationiques contenant des lieurs obtenus a partir d'acides amines neutres ou charges positivement
EP1019361A1 (fr) Cytofectines cationiques polyfonctionnelles, formulations et procede de generation de complexes actifs de transfection cytofectine:polynucleotide
AU736143B2 (en) Cationic amphiphile compositions for intracellular delivery of therapeutic molecules
AU716706C (en) Cationic amphiphiles and plasmids for intracellular delivery of therapeutic molecules
Bennett Design, synthesis, and formulation of cationic lipids as carriers of plasmid DNA for In vitro and In vivo cell transfection
AU5718099A (en) Cationic amphiphiles and plasmids for intracellular delivery of therapeutic molecules

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: C2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 37 AND 38, CLAIMS, REPLACED BY NEW PAGES 37 AND 38; PAGES 1/16-16/16, DRAWINGS, REPLACED BY NEW PAGES 1/16-16/16; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase