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WO1998051285A2 - Formulations amphiphiles cationiques - Google Patents

Formulations amphiphiles cationiques Download PDF

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Publication number
WO1998051285A2
WO1998051285A2 PCT/US1998/009974 US9809974W WO9851285A2 WO 1998051285 A2 WO1998051285 A2 WO 1998051285A2 US 9809974 W US9809974 W US 9809974W WO 9851285 A2 WO9851285 A2 WO 9851285A2
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WIPO (PCT)
Prior art keywords
peg
derivative
amphiphile
lipid
biologically active
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PCT/US1998/009974
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English (en)
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WO1998051285A3 (fr
Inventor
Jennifer D. Tousignant
John Marshall
Simon J. Eastman
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Genzyme Corporation
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Priority to AU74900/98A priority Critical patent/AU7490098A/en
Publication of WO1998051285A2 publication Critical patent/WO1998051285A2/fr
Publication of WO1998051285A3 publication Critical patent/WO1998051285A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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
    • 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
    • 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 novel formulations of cationic amphiphilic compounds that facilitate the intracellular delivery of biologically active (therapeutic) molecules.
  • the novel formulations of cationic amphiphiles are particularly useful in relation to treating disease states such as by gene therapy.
  • immunoglobin proteins (2) polynucleotides such as genomic DNA, cDNA, or mRNA
  • Examples of diseases that it is hoped can be treated by gene therapy include:
  • cystic fibrosis inherited disorders such as cystic fibrosis, Gaucher's disease, Fabry's disease, and
  • muscular dystrophy Representative of acquired disorders that can be treated are:
  • small cell lung cancer (2) for cardiovascular conditions — progressive heart failure, restenosis, and hemophilias; and (3) for neurological conditions — traumatic brain injury.
  • Transfection requires successful transfection of target cells in a patient.
  • Transfection may generally be defined as the process of introducing an expressible polynucleotide (for example a gene, a cDNA, or an mRNA) into a cell.
  • an expressible polynucleotide for example a gene, a cDNA, or an mRNA
  • Successful expression of the encoding polynucleotide leads to production in the cells of a normal protein and leads to correction of the disease state associated with the abnormal gene. Therapies based on providing such proteins directly to target cells
  • Cystic fibrosis a common lethal genetic disorder, is a particular example of a disease that is a target for gene therapy.
  • the disease is caused by the presence of one or more mutations in the gene that encodes a protein known as cystic fibrosis transmembrane conductance regulator ("CFTR"), and which regulates the movement of ions (and therefore fluid) across the cell membrane of epithelial cells, including lung epithelial cells.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Abnormal ion transport in airway cells leads to abnormal mucous secretion, inflammation and infection, tissue damage, and eventually death.
  • amphiphiles Compounds having both such domains may be termed amphiphiles, and many lipids and synthetic lipids that have been disclosed for use in facilitating such intracellular delivery (whether for in vitro or in vivo application) meet this definition.
  • amphiphiles One particularly important class of such amphiphiles is the cationic amphiphiles.
  • cationic amphiphiles In general, cationic amphiphiles have polar groups that are capable of being positively charged at or around physiological pH,
  • amphiphiles interact with the many types of biologically active (therapeutic) molecules including, for example, negatively charged polynucleotides such as DNA.
  • cationic amphiphilic compounds that have both polar and non- polar domains and that are stated to be useful in relation to intracellular delivery of biologically active molecules are found, for example, in the following references, which contain also useful discussion of (1) the properties of such compounds that are understood in the art as making them suitable for such applications, and (2) the nature of structures, as understood in the art, that are formed by complexing of such amphiphiles with therapeutic molecules intended for intracellular delivery.
  • DOTMA N-[1(2,3-dioleyloxy)propyl]- N,N,N-trimethylammonium chloride
  • This invention provides for formulations of cationic amphiphiles that are particularly effective to facilitate transport of biologically active molecules, such as a polynucleotide, into cells.
  • the formulations comprise a derivative of polyethylene glycol (PEG), and one or more cationic amphiphiles, and optionally a neutral co-lipid.
  • PEG polyethylene glycol
  • a derivative of PEG is any hydrophobic group attached to the PEG polymer.
  • suitable PEG derivatives are polyethylene glycol 5000 — dimyristoylphosphatidylethanolamine (hereinafter PEG (5000) — DMPE), and polyethylene glycol 2000 — dimyristoylphosphatidylethanolamine (hereinafter PEG ⁇ 2000) — DMPE).
  • the formulation is then mixed with a biologically active molecule such as a polynucleotide, to form a therapeutic composition in which the cationic amphiphile and polynucleotide are in intimate contact.
  • a biologically active molecule such as a polynucleotide
  • the PEG derivative stabilizes the therapeutic composition by preventing undesired further aggregation of the cationic amphiphile/polynucleotide complexes.
  • compositions include: (1) cationic amphiphile/polynucleotide complexes may be maintained at higher concentrations in solution, and (2) such compositions may be more efficiently delivered by aerosol to the lung.
  • neutral co-lipids that are useful in the practice of the invention are diphytanoylphosphatidylethanolamine and dioleoyl
  • DOPE phosphatidylethanolamine
  • cationic amphiphiles that are useful in the practice of the invention are:
  • the invention provides a method for facilitating the transfer of biologically active molecules into cells comprising the steps of: preparing a
  • dispersion of one or more cationic amphiphiles, a neutral co-lipid, and a PEG - derivative mixing said dispersion with a biologically active molecule to form a complex between said amphiphile and said biologically active molecule; and contacting cells with said complex, thereby facilitating transfer of said biologically- active molecule into the cells.
  • a derivative of polyethylene glycol is used to stabilize, and to enhance the transfecting properties of, therapeutic compositions than comprise a cationic amphiphile. According to the practice of the present invention it has been surprisingly determined that the stability and
  • transfection-enhancing capability of cationic amphiphile/neutral co-lipid compositions can be substantially improved by adding to such formulations small additional amounts of one or more derivatized polyethylene glycol compounds.
  • Such enhanced performance is particularly apparent whether measured by stability of cationic amphiphile/co-lipid formulations to storage and manipulation, including in
  • polyethylene glycol useful in the practice of the invention include numerous phospholipid conjugates of polyethylene glycol. With respect to the design of such derivatives, selection of certain phospholipids is preferred, as is the selection of particular polyethylene glycol polymers.
  • the structure of a typical derivative is provided in Figure 1.
  • phospholipid for inclusion therein, species of phosphatidylethanolamine are preferred. It is preferred that the fatty acid(acyl) chains thereof be selected from the group consisting of C 10 , C 12 , and C 14 , most
  • Each phospholipid contains 2 fatty acid chains, and it is within the practice of the invention to provide as phospholipid component, species having 2 different acyl chains.
  • Two highly preferred species thereof include dimyristoylphosphatidylethanolamine (di C 14 ) (“DMPE”) and dilaurylphosphatidylethanolamine (di C 12 ) (“DLPE”).
  • DMPE dimyristoylphosphatidylethanolamine
  • DLPE dilaurylphosphatidylethanolamine
  • DLPE dilaurylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DSPE distearoylphosphatidylethanoiamine
  • the polymer be linear, having a molecular weight from about 1 ,000 and 10,000, preferred species thereof including those having molecular weights from about 1500 to 7000, with 2000 and 5000 being examples of useful, and commercially available sizes.
  • the molecular weight assigned to PEG in such products often represents a molecular weight average, there being shorter and longer molecules in the product.
  • Such molecular weight ranges are typically a consequence of the synthetic procedures used, and the use of any such product is within the practice of the invention as long as a substantial fraction of polymer population falls within or near the above-suggested MW range of 1000-10,000.
  • derivatized-PEG species that (1) include more than one attached phospholipid, or (2) include branched PEG sequence, or (3) include both of modifications (1) and (2).
  • preferred species of derivatized PEG include
  • polyethylene glycol 5000-dimyristoylphosphatidylethanolamine also referred to as PEG (5000) — DMPE;
  • polyethylene glycol 5000-dilaurylphosphatidylethanolamine also referred to as PEG (5000) — DLPE
  • Certain phospholipid derivatives of PEG may be obtained from commercial suppliers.
  • the following species di C14:0, di C16:0, di C18:0, di C18:1 , and 16:0/18:1 are available as average 2000 or average 5000 MW PEG derivatives from Avanti Polar Lipids, Alabaster, AL, USA, as catalog nos. 880150, 880160, 880120, 880130, 880140, 880210, 880200, 880220, 880230, and 880240.
  • neutral co-lipids with cationic amphiphiles substantially enhances transfection capability.
  • Representative neutral co-lipids include dioleoylphosphatidylethanolamine ("DOPE"), the species most commonly used in the art, diphytanoylphosphatidylethanoiamine, lyso-phosphatidylethanolamines other
  • phosphatidyl-ethanolamines phosphatidylcholines, lyso-phosphatidylchoiines and cholesterol.
  • a preferred molar ratio of cationic amphiphile to colipid is about 1 :1.
  • preferred formulations may also be defined in relation to mole percent of PEG derivative.
  • PEG derivative for example, with respect to a
  • formulation of cationic amphiphile/neutral co-lipid/PEG derivative that is 1 :2: 0.05 on a molar basis, the derivative is present at about 0.05/3.05 x100 or 1.6 mole percent.
  • use of formulations ranging from about 0.1 to 10 mole percent of PEG-derivative is preferred, with formulations of about 0.5 to
  • a representative preferred formulation according to the practice of the present invention has a cationic amphiphile : neutral colipid: PEG-derivative molar composition ratio of about 1:2:0.125.
  • Another representative formulation has a cationic amphiphile : neutral co-lipid : PEG-derivative molar composition ratio of about 1 :2:0.5.
  • the neutral co-lipid is diphytanoylphosphatidylethanolamine, or is DOPE
  • the PEG derivative is a DMPE or DLPE conjugate of PEG 2000 or PEG 5000 .
  • the neutral co-lipid is diphytanoylphosphatidyl ethanolamine
  • the PEG derivative is
  • amphiphiles are (see the WO 96/18372 publication):
  • amphiphile No. 67 having the IUPAC name (3-Amino-propyl)-[4-(3-amino- propylamino)-butyl]-carbamic acid 17-(1 ,5-dimethyl-hexyl)-10J3-dimethyl- 2,3,4,7,8,9,10,11 ,12,13,14,15,16,17-tetradecahydro-1 H-cyclopenta[a]phenanthren- 3-yl ester; and
  • amphiphile No. 53 having the IUPAC name (4-Amino-butyl)-(3-amino-propyl)- carbamic acid 17-(1 ,5-dimethyl-hexyl)-10,13-dimethyl-
  • compositions of the present invention may or may not contain such transacylation byproducts, or other byproducts, and that the presence of such byproducts does not prevent the therapeutic use of the compositions containing them. Rather use of such compositions is within the practice of the invention, and such compositions and the novel molecular species thereof are therefore specifically claimed.
  • the present invention provides for pharmaceutical compositions that facilitate
  • compositions of the invention facilitate entry of biologically active molecules into tissues and organs such as the gastric mucosa, heart, lung, and solid tumors. Additionally, compositions of the invention facilitate entry of biologically active molecules into cells that are maintained in vitro, such as in tissue culture.
  • the amphiphilic nature of the compounds of the invention enables them to associate with the lipids of ceil membranes, other cell surface molecules, and tissue surfaces, and to fuse or to attach thereto.
  • One type of structure that can be formed by amphiphiles is the liposome, a vesicle formed into a more or less spherical bilayer, that is stable in biological fluids and can entrap biological molecules targeted for intracellular delivery.
  • liposomal compositions permit biologically active molecules carried therewith to gain access to the interior of a cell through one or more cell processes including endocytosis and pinocytosis.
  • the cationic amphiphiles of the invention need not form highly organized vesicles in order to be effective, and in fact can assume (with the biologically active molecules to which they bind) a wide variety of loosely organized structures. Any of such structures can be present in pharmaceutical preparations of the invention and can contribute to the effectiveness thereof.
  • amphiphiles of the invention include: (a) polynucleotides such as genomic DNA, cDNA, and mRNA that encode for therapeutically useful proteins as
  • antisense polynucleotides whether RNA or DNA, that are useful to inactivate transcription products of genes and which are useful, for example, as therapies to regulate the growth of malignant cells;
  • vesicles or other structures formed from numerous of the cationic amphiphiles are not preferred by those skilled in the art in order to deliver low molecular weight biologically active molecules. Although not a preferred embodiment of the present invention, it is nonetheless within the practice of the invention to deliver such low molecular weight molecules intracellularly.
  • Representative of the types of low molecular weight biologically active molecules that can be delivered include
  • Cationic amphiphile species of the invention may be blended so that two or more species thereof are used, in combination, to facilitate entry of biologically active molecules into target cells and/or into subcellular compartments thereof Cationic amphiphiles of the invention can also be blended for such use with
  • amphiphiles that are known in the art.
  • Dosages of the pharmaceutical compositions of the invention will vary, depending on factors such as half-life of the biologically-active molecule, potency of the biologically-active molecule, half-life of the amphiphile(s), any potential adverse effects of the amphiphile(s) or of degradation products thereof, the route of administration, the condition of the patient, and the like. Such factors are capable of determination by those skilled in the art.
  • compositions of the invention A variety of methods of administration may be used to provide highly accurate dosages of the pharmaceutical compositions of the invention.
  • Such preparations can be administered orally, parenterally, topically, transmucosally, or by injection of a preparation into a body cavity of the patient, or by using a sustained-release formulation containing a biodegradable material, or by onsite delivery using additional micelles, gels and liposomes.
  • Nebulizing devices, powder inhalers, and aerosolized solutions are representative of methods that may be used to administer such preparations to the respiratory tract.
  • compositions of the invention can in general be formulated with excipients (such as the carbohydrates lactose, trehalose, sucrose, mannitol, maltose or galactose, and inorganic or organic salts) and may also be lyophilized (and then rehydrated) in the presence of such excipients prior to use.
  • excipients such as the carbohydrates lactose, trehalose, sucrose, mannitol, maltose or galactose, and inorganic or organic salts
  • Conditions of optimized formulation for each amphiphile of the invention are capable of determination by those skilled in the pharmaceutical art.
  • a principal aspect of the invention involves providing a composition that comprises a biologically active molecule (for example, a polynucleotide) and one or more cationic amphiphiles (including optionally one or more co-lipids), and then maintaining said composition in the presence of one or more excipients as aforementioned, said resultant composition being in liquid or
  • an additional and valuable characteristic of the amphiphiles of the invention is that any such potentially adverse effect can be minimized owing to the greatly enhanced in vivo activity of the amphiphiles of the invention in comparison with amphiphilic compounds known in the art. Without being limited as to theory, it is believed that osmotic stress (at low total solute concentration) may contribute positively to the successful transfection of polynucleotides into cells in vivo.
  • Such a stress may occur when the pharmaceutical composition, provided in unbuffered water, contacts the target cells.
  • Use of such otherwise preferred compositions may therefore be incompatible with treating target tissues that already are stressed, such as has damaged lung tissue of a cystic fibrosis patient. Accordingly, and using sucrose as an example, selection of concentrations of this excipient that range from about 15 mM to about 200 mM provide a compromise between the goals of (1) stabilizing the pharmaceutical composition to storage and (2) minimizing any effects that high concentrations of solutes in the composition may have on transfection performance.
  • formulations is subject to experimentation, but can be determined by those skilled in the art for each such formulation.
  • An additional aspect of the invention concerns the protonation state of the cationic amphiphiles of the invention prior to their contacting plasmid DNA in order to form a therapeutic composition, or prior to the time when said therapeutic composition contacts a biological fluid. It is within the practice of the invention to provide fully protonated, partially protonated, or free base forms of the amphiphiles in order to form, or maintain, such therapeutic compositions. Examples
  • amphiphile No. 53 and the neutral lipid dioleoylphosphatidylethanolamine (“DOPE”) were each dissolved in chloroform as stock preparations. Following combination of the solutions (as a 1 :1 molar composition), a thin film was produced by removing chloroform from the mixture by evaporation under reduced pressure (20 mm Hg). The film was further dried under vacuum (1 mm Hg) for 24 hours.
  • some of the amphiphiles of the invention participate in transacylation reactions with co-lipids such as DOPE, or are subject to other reactions which may cause decomposition thereof. Accordingly, it is preferred that amphiphile/co-lipid compositions be stored at low temperature, such as -70 degrees C under inert gas, until use.
  • the lipid film was then hydrated with sterile deionized water (1 ml) for 10 minutes, and then vortexed for 2 minutes (sonication for 10 to 20 seconds in a bath sonicator may also be used, and sonication has proved useful for other amphiphiles such as DC-chol).
  • the resulting suspension was then diluted with 4 ml of water to yield a solution that is 670 ⁇ M in cationic amphiphile and 670 ⁇ M in neutral colipid. Similar experiments were also performed using other amphiphiles of the invention. With respect to amphiphile No. 67, the optimum molar ratio of amphiphile to DOPE under the conditions tested was determined to be 1 :2, not 1:1. Optimized ratios for any of the amphiphiles of the invention can be determined by following, generally, the procedures described herein.
  • pCMV ⁇ ClonTech., Palo Alto, CA
  • OptiMEM culture medium Gibco/ BRL No. 31885-013
  • the resulting solution had a DNA concentration of 960 ⁇ M (assuming an average molecular weight of 330 daltons for nucleotides in the encoding DNA).
  • the construct pCF1- ⁇ (described below) may also be used and generally provides about a 2-fold enhancement over pCMV ⁇ .
  • DNA solutions (165 ⁇ l, 960 ⁇ M) were pipetted into 8 wells containing OptiMEM (165 ⁇ l), and the resulting 480 ⁇ M solutions were then serially diluted 7 times to generate 8 separate 165 ⁇ l solutions from each well, with the concentrations of DNA in the wells ranging from 480 ⁇ M to 3.75 ⁇ M.
  • the 64 test solutions (cationic amphiphile: neutral lipid) were then combined with the 64 DNA solutions to give separate mixtures in 64 wells, each having a volume of 330 ⁇ l, with DNA concentrations ranging from 240 ⁇ M to 1.875 ⁇ M along one axis, and lipid concentrations ranging from 167 ⁇ M to 1.32 ⁇ M along the other axis.
  • 64 solutions were prepared in all, each having a different amphiphile: DNA ratio and/or concentration.
  • the solutions of DNA and amphiphile were allowed to stand for 15 to 30 minutes in order to allow complex formation.
  • a CFT-1 cell line (human cystic fibrosis bronchial epithelial cells immortalized with transforming proteins from papillomavirus) provided by Dr. James Yankaskas, University of North Carolina, Chapel Hill, was used for the in vitro assay.
  • the cells are homozygous for a mutant allele (deletion of phenylalanine at position 508, hereinafter ⁇ F508 ) of the gene encoding for cystic fibrosis transmembrane conductance regulator ("CFTR") protein.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • CFTR cystic fibrosis transmembrane conductance regulator
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Mutation of the CFTR gene results typically in complete loss (or at least substantial impairment) of Cl ⁇ channel activity across, for example, cell membranes of affected epithelial tissues.
  • the ⁇ F508 mutation is the most common mutation associated with cystic fibrosis disease.
  • the cells were cultured in Hams F12 nutrient media (Gibco/BRL No. 31765- 027) supplemented with 2% fetal bovine serum ("FBS", Irvine Scientific, No. 3000) and 7 additional supplements. Cells were then plated into 96-well tissue culture
  • the 100 ⁇ l solutions of DNA-lipid complex were maintained over the cells for 6 hours, after which 50 ⁇ l of 30% FBS (in OptiMEM) was added to each well. After a further 20-hour incubation period, an additional 100 ⁇ l of 10% FBS in OptiMEM was also added. Following a further 24-hour incubation period, cells were assayed for expression of protein and ⁇ -galactosidase.
  • the resultant medium was removed from the plates and the cells washed with phosphate buffered saline. Lysis buffer (50 ⁇ l, 250 mM Tris-HCI, pH 8.0, 0.15% Triton X-100) was then added, and the cells were lysed for 30 minutes. The 96-well plates were carefully vortexed for 10 seconds to dislodge the cells and cell debris, and 5 ⁇ l volumes of lysate from each well were transferred to a plate containing 100 ⁇ l volumes of Coomassie Plus® protein assay reagent (Pierce Company, No. 23236). The protein assay plates were read by a Bio-Rad Model 450 plate-reader containing a 595 nm filter, with a protein standard curve included in every assay.
  • Lysis buffer 50 ⁇ l, 250 mM Tris-HCI, pH 8.0, 0.15% Triton X-100
  • the level of ⁇ -galactosidase activity in each well was measured by adding phosphate buffered saline (50 ⁇ i) to the remaining lysates, followed by addition of a buffered solution consisting of chlorophenol red galactopyranoside (100 ⁇ l, 1 mg per ml, Calbiochem No. 220588), 60 mM disodium hydrogen phosphate pH 8.0, 1 mM magnesium sulfate, 10 mM potassium chloride, and optionally 50 mM 2- mercaptoethanol.
  • phosphate buffered saline 50 ⁇ i
  • a buffered solution consisting of chlorophenol red galactopyranoside (100 ⁇ l, 1 mg per ml, Calbiochem No. 220588), 60 mM disodium hydrogen phosphate pH 8.0, 1 mM magnesium sulfate, 10 mM potassium chloride, and optionally 50 mM 2- mercaptoethanol.
  • the chlorophenol red galactopyranoside following enzymatic ( ⁇ - galactosidase) hydrolysis, gave a red color which was detected by a plate-reader containing a 570 nm filter.
  • a ⁇ -galactosidase (Sigma No. G6512) standard curve was included to calibrate every assay.
  • optical data determined by the plate-reader allowed determination of ⁇ -galactosidase activity and protein content.
  • DMRIE diimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium
  • compounds of the invention are particularly effective in transfecting airway epithelial cells and inducing therein ⁇ -galactosidase expression.
  • This assay was used to assess the ability of the cationic amphiphiles of the invention to transfect cells in vivo from live specimens.
  • the lungs of balb/c mice were instilled intra-nasally (the procedure can also be performed trans- tracheally) with 100 ⁇ l of cationic amphiphile No. 53:DNA complex, which was allowed to form during a 15-minute period prior to administration according to the
  • amphiphile premixed with co-lipid, see below
  • DNA encoding the reporter (CAT) gene was diluted with water to a concentration twice the
  • lipid was gently combined with the DNA (in a polystyrene tube), and the complex allowed to form for 15 minutes before the mice were instilled therewith (the lipid and DNA are both warmed to 30°C for 5 minutes prior to mixing and maintained at 30 °C during the 15 minutes of complex formation to reduce the
  • pCF1/CAT The plasmid used, pCF1/CAT (see Example 4, pages 82-85, and Figure 18A of international patent publication WO 96/18372 published June 20, 1996), provides an encoding DNA for chloramphenicol acetyl transferase enzyme.
  • mice Two days following transfection, mice were sacrificed, and the lungs and trachea removed, weighed, and homogenized in a buffer solution (250 mM Tris, pH 7.8, 5mM EDTA). The homogenate was clarified by centrifugation, and the deacetylases therein were inactivated by heat treatment at 65°C for twenty minutes. Lysate was incubated for thirty minutes with acetyl coenzyme A and C 14" chloramphenicol (optimum times vary somewhat for the different amphiphile species of the invention). CAT enzyme activity was then visualized by thin layer chromatography ("TLC") following an ethyl acetate extraction. Enzyme activity was
  • the presence of the enzyme CAT will cause an acetyl group to be transferred from acetylcoenzyme A to C 1 -chloramphenicol.
  • the acetylated/radiolabeled chloramphenicol migrates faster on a TLC plate and thus its presence can be detected.
  • the amount of CAT that had been necessary to generate the determined amount of acetylated chloramphenicol can then be calculated from standards.
  • amphiphile No.53 The activity of amphiphile No.53 was determined in the CAT assay in relation to the recognized transfection reagents DMRIE and DC-Choi. Enhanced ability of the No. 53 amphiphile (measured as ng CAT activity per 100 mg lung tissue) to
  • transfect cells in vivo was determined in relation to DMRIE.
  • the biologically active macromolecule is an encoding DNA.
  • novel vectors plasmids
  • pCF1 contains the enhancer/promoter region from the immediate early gene of cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • the vector also contains a drug-resistance marker that encodes the aminoglycosidase 3'-phosphotransferase gene (derived from the transposon Tn903, A. Oka et al., Journal of Molecular Biolo ⁇ v. 147, 217-226, 1981) thereby conferring resistance to kanamycin. Further details of pCF1 structure are provided directly below, including description of placement therein of a cDNA sequence encoding for cystic fibrosis transmembrane conductance regulator (CFTR) protein.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the pCF1 vector is based on the commercially available vector pCMV ⁇ (Clontech).
  • the pCMV ⁇ construct has a pUC19 backbone (J. Vieira, et al., Gene, 19, 259-268, 1982) that includes a prokaryotic origin of replication derived originally from pBR322.
  • pCFI-plasmid (as constructed to include a nucleotide sequence coding for CFTR) are as follows. Proceeding clockwise the human cytomegalovirus immediate early gene promoter and enhancer, a fused tripartite leader from adenovirus and a hybrid intron, a linker sequence, the CFTR cDNA, an additional linker sequence, the bovine growth hormone polyadenylation signal, pUC origin of replication and backbone, and the kanamycin resistance gene. The pCF1- CFTR plasmid has been completely sequenced on both strands.
  • the human cytomegalovirus immediate early gene promoter and enhancer spans the region from nucleotides 1-639. This corresponds to the region from -522
  • the CAAT box is located at nucleotides 486-490 and the TATA box is at nucleotides 521-525 in pCF1-CFTR.
  • the CFTR transcript is predicted to initiate at nucleotide 548, which is the transcriptional start site of the CMV promoter.
  • the hybrid intron is composed of a fused tri-partite leader from adenovirus containing a 5' splice donor signal, and a 3' splice acceptor signal derived from an IgG gene.
  • the elements in the intron are as follows: the first leader (nucleotides 705-745), the second leader (nucleotides 746-816), the third leader (partial, nucleotides 817-877), the splice donor sequence and intron region from the first leader (nucleotides 878-1042), and the mouse immunoglobulin gene splice donor
  • GT) is at nucleotides 887- 888
  • G) is at nucleotides 1128-1129
  • intron is 230 nucleotides.
  • the CFTR coding region comprises nucleotides 1183- 5622.
  • nucleotides of the 3' untranslated region of the CFTR cDNA 21 nucleotides of linker sequence and 114 nucleotides of the BGH poly A signal.
  • the BGH poly A signal contains 90 nucleotides of flanking sequence 5' to the conserved AAUAAA and 129 nucleotides of flanking sequence 3' to the AAUAAA motif.
  • the primary CFTR transcript is predicted to be cleaved downstream of the BGH polyadenylation signal at nucleotide 5808.
  • a recommended procedure for formulating and using the pharmaceutical compositions of the invention to treat cystic fibrosis in human patients is as follows.
  • a thin film (evaporated from chloroform) can be produced wherein amphiphile No. 53 and DOPE are present in the molar ratio of 1 :1 [Alternatively, the chloroform can be removed in a dessicator placed in a cooling bath at approximately -10 degrees C under high vacuum, for example 1 mm Hg, for 12-24 hours].
  • the amphiphile- containing film is then rehydrated in water-for -injection with gentle vortexing to a resultant amphiphile concentration of about 3mM.
  • a Puritan Bennett Raindrop nebulizer from a Puritan Bennett Raindrop nebulizer from a homogeneous phase
  • amphiphile-containing film it may be advantageous to prepare the amphiphile-containing film to include also one or more further ingredients that act to stabilize the final amphiphile/DNA composition. Accordingly, it may be preferred to prepare the amphiphile-containing film using an additional ingredient, PEG ⁇ 5000) - DMPE.
  • PEG ⁇ 5000 polyethylene glycol 5000- dimyristoylphoshatidyl ethanolamine
  • Additional fatty acid species of PEG-PE may be used in
  • PEG (5000) -DMPE is believed to stabilize the therapeutic compositions by preventing further aggregation of formed amphiphile/DNA complexes. Additional discussion of the use of these ingredients in found in aforementioned WO 96/18372 at, for example, page 87.
  • pCFI-CFTR plasmid (containing an encoding sequence for human cystic fibrosis transmembrane conductance regulator, see Example 4) is provided in water- for-injection at a concentration, measured as nucleotide, of 4 mM. Complexing of the plasmid and amphiphile is then allowed to proceed by gentle contacting of the
  • aerosolized DNA it is presently preferred to deliver aerosolized DNA to the lung at a concentration thereof of between about 2 and about 12 mM (as nucleotide).
  • a sample of about 10 to about 40 ml is generally sufficient for one aerosol administration to the lung of an adult patient who is homozygous for the ⁇ F508 mutation in the CFTR-encoding gene.
  • cationic amphiphiles of the present invention are substantially more effective — in vivo — than other presently available amphiphiles, and thus may be used at substantially lower concentrations than known cationic amphiphiles. There results the opportunity to substantially minimize side effects (such as amphiphile toxicity, inflammatory response) that would
  • amphiphiles of the invention were designed so that the metabolism thereof would rapidly proceed toward relatively harmless biologically-compatible components.
  • compositions may be preferable to avoid use of chloroform when the cationic amphiphile and the colipid are prepared together.
  • An alternate method to produce such compositions may
  • the cationic amphiphile, the neutral co-lipid DOPE, and PEG (5000) -DMPE are weighed into vials, and each is dissolved in t-butanol:water 9:1 with vortexing, followed by transfer to a single volumetric flask. An appropriate amount of each lipid is selected to obtain a molar ratio of cationic amphiphile to DOPE to DMPE-PEG of 1:2: 0.05.
  • the resultant solution is then vortexed, and further diluted as needed with t-butanol:water 9:1 , to obtain the desired concentration.
  • the solution is then filtered using a sterile filter (0.2 micron, nylon).
  • One mL of the resultant filtered 1:2: 0.05 solution is then pipetted into individual vials.
  • the vials are partially stoppered with 2-leg butyl stoppers and placed on a tray for lyophilization.
  • the t-butanol:water 9:1 solution is removed by freeze drying over 2 to 4 days at a temperature of approximately -5°C.
  • the lyophilizer is then backfilled with argon that is passed through a sterile 0.2 micron filter.
  • the stoppers are then fully inserted into the vials, and the vials are then crimped shut with an aluminum crimp-top.
  • the vials are then maintained at -70°C
  • An alternate lyophilization procedure is as follows. 4 mL of stock solution (amphiphile No. 67: DOPE: PEG ( 5000 )-DMPE as 1:2: 0.05) in tBuOH:dH 2 O (9:1) was placed in a 20 mL serum vial and partially stoppered with a 2-leg butyl stopper. The vials were placed on a precooled shelf (-30 degrees C) in a tray lyophilizer and allowed to cool for 30 minutes. Vacuum was applied and the samples were held at approximately 200 mTorr for 1 hour. The shelf temperature was then raised to -5 degrees C for 8 hours, and then the shelf temperature was raised to 20 degrees C.
  • the samples were then allowed to lyophilize to dryness (about 16 hours).
  • the lyophilizer was backfilled with filtered argon gas and the samples were stoppered.
  • the vials were sealed with an aluminum crimp top and stored at -80 degrees C.

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Abstract

Cette invention se rapporte à de nouvelles formulations d'amphiphiles cationiques qui facilitent le transport de molécules biologiquement actives (thérapeutiques) à l'intérieur de cellules. Ces formulations comportent un ou plusieurs amphiphiles cationiques, un co-lipide neutre tel qu'une diphytanoylphosphatidyléthanolamine, et un ou plusieurs dérivés de polyéthylène glycol. Les molécules thérapeutiques que l'on peut transporter à l'intérieur de cellules, conformément à cette invention, contiennent de l'ADN, de l'ARN et des polypeptides. Les utilisations possibles des formulations thérapeutiques de cette invention incluent, entre autres, certaines thérapies géniques et l'administration à des cellules de polynucléotides antisens ou de polypeptides biologiquement actifs.
PCT/US1998/009974 1997-05-15 1998-05-15 Formulations amphiphiles cationiques WO1998051285A2 (fr)

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WO1999052858A2 (fr) * 1998-04-08 1999-10-21 Celltech Therapeutics Limited Lipides
WO2000062813A2 (fr) * 1999-04-20 2000-10-26 The University Of British Columbia Lipides peg cationiques et méthodes d'utilisation
WO2001080900A2 (fr) * 2000-04-20 2001-11-01 The University Of British Columbia Procedes permettant d'ameliorer la transfection a mediation splp (particule plasmide-lipide stabilisee) au moyen de destabilisateurs de la membrane endosomale
WO2001015755A3 (fr) * 1999-09-01 2002-02-28 Genecure Pte Ltd Procedes et compositions d'administration d'agents pharmaceutiques
US6852334B1 (en) 1999-04-20 2005-02-08 The University Of British Columbia Cationic peg-lipids and methods of use
WO2005026372A1 (fr) * 2003-09-15 2005-03-24 Protiva Biotherapeutics, Inc. Composes conjugues lipidiques polyethyleneglycol-dialkyloxypropyle et utilisations de ces composes
EP1758595A2 (fr) * 2004-03-02 2007-03-07 Yissum Research Development Company of the Hebrew University of Jerusalem Utilisation de conjugues lipidiques dans le traitement de maladies
US7189705B2 (en) 2000-04-20 2007-03-13 The University Of British Columbia Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers
US7772196B2 (en) 2000-01-10 2010-08-10 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
US7811999B2 (en) 2000-01-10 2010-10-12 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Use of lipid conjugates in the treatment of diseases
US7893226B2 (en) 2004-09-29 2011-02-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Use of lipid conjugates in the treatment of diseases
US7982027B2 (en) 2003-07-16 2011-07-19 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering RNA
US8017804B2 (en) 2004-05-05 2011-09-13 Silence Therapeutics Ag Lipids, lipid complexes and use thereof
US8076312B2 (en) 2000-01-10 2011-12-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Use of lipid conjugates in the treatment of disease
US8232256B2 (en) 2006-07-21 2012-07-31 Silence Therapeutics Ag Means for inhibiting the expression of protein kinase 3
US8304395B2 (en) 2000-01-10 2012-11-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Lipid conjugates in the treatment of disease
US8372815B2 (en) 2000-01-10 2013-02-12 Yissum Research Development Company Use of lipid conjugates in the treatment of conjunctivitis
US8492359B2 (en) 2008-04-15 2013-07-23 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US8501701B2 (en) 2000-01-10 2013-08-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Use of lipid conjugates in the treatment of disease
US8569256B2 (en) 2009-07-01 2013-10-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US8852472B2 (en) 2004-12-27 2014-10-07 Silence Therapeutics Gmbh Coated lipid complexes and their use
US8859524B2 (en) 2005-11-17 2014-10-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Lipid conjugates in the treatment of chronic rhinosinusitis
US8865681B2 (en) 2004-03-02 2014-10-21 Yissum Research Development Company of the Hebrew Unitersity of Jerusalem Use of lipid conjugates in the treatment of diseases or disorders of the eye
US8883761B2 (en) 2001-01-10 2014-11-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases associated with vasculature
US8906882B2 (en) 2005-11-17 2014-12-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Lipid conjugates in the treatment of allergic rhinitis
US8916539B2 (en) 2000-01-10 2014-12-23 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of disease
US9006417B2 (en) 2010-06-30 2015-04-14 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US9040078B2 (en) 2000-01-10 2015-05-26 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases of the nervous system
US9181545B2 (en) 2004-06-07 2015-11-10 Protiva Biotherapeutics, Inc. Lipid encapsulating interfering RNA
US9492386B2 (en) 2002-06-28 2016-11-15 Protiva Biotherapeutics, Inc. Liposomal apparatus and manufacturing methods
US9878042B2 (en) 2009-07-01 2018-01-30 Protiva Biotherapeutics, Inc. Lipid formulations for delivery of therapeutic agents to solid tumors
US11013811B2 (en) 2009-05-11 2021-05-25 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Lipid-polymer conjugates, their preparation and uses thereof
US11591544B2 (en) 2020-11-25 2023-02-28 Akagera Medicines, Inc. Ionizable cationic lipids
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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9018187B2 (en) 2009-07-01 2015-04-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018372A2 (fr) * 1994-12-09 1996-06-20 Genzyme Corporation Amphiphiles cationiques et plasmides destines a la liberation intracellulaire de molecules therapeutiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018372A2 (fr) * 1994-12-09 1996-06-20 Genzyme Corporation Amphiphiles cationiques et plasmides destines a la liberation intracellulaire de molecules therapeutiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
E.K. WASAN ET AL.: "plasmid dna is protected against ultrasonic cavitation-induced damage when complexed to cationic liposomes" JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 85, no. 4, April 1996, pages 427-433, XP000558752 washington (us) *

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WO1999052858A3 (fr) * 1998-04-08 2000-04-06 Celltech Therapeutics Ltd Lipides
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US6583301B1 (en) 1998-04-08 2003-06-24 Celltech R & D Limited Lipids
US7531676B2 (en) 1998-04-08 2009-05-12 Celltech R & D Limited Lipids
WO2000062813A2 (fr) * 1999-04-20 2000-10-26 The University Of British Columbia Lipides peg cationiques et méthodes d'utilisation
WO2000062813A3 (fr) * 1999-04-20 2001-08-09 Univ British Columbia Lipides peg cationiques et méthodes d'utilisation
US6852334B1 (en) 1999-04-20 2005-02-08 The University Of British Columbia Cationic peg-lipids and methods of use
AU781684B2 (en) * 1999-09-01 2005-06-09 Genecure Pte Ltd Methods and compositions for delivery of pharmaceutical agents
WO2001015755A3 (fr) * 1999-09-01 2002-02-28 Genecure Pte Ltd Procedes et compositions d'administration d'agents pharmaceutiques
US7709457B2 (en) 1999-09-01 2010-05-04 Genecure Pte Ltd. Methods and compositions for delivery of pharmaceutical agents
US7320963B2 (en) 1999-09-01 2008-01-22 Genecure Pte Ltd Methods and compositions for delivery of pharmaceutical agents
US8076312B2 (en) 2000-01-10 2011-12-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Use of lipid conjugates in the treatment of disease
US8304395B2 (en) 2000-01-10 2012-11-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Lipid conjugates in the treatment of disease
US8501701B2 (en) 2000-01-10 2013-08-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Use of lipid conjugates in the treatment of disease
US9040078B2 (en) 2000-01-10 2015-05-26 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases of the nervous system
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WO2001080900A2 (fr) * 2000-04-20 2001-11-01 The University Of British Columbia Procedes permettant d'ameliorer la transfection a mediation splp (particule plasmide-lipide stabilisee) au moyen de destabilisateurs de la membrane endosomale
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