WO2001042200A1 - Cationic amphiphiles for intracellular delivery of therapeutic molecules - Google Patents
Cationic amphiphiles for intracellular delivery of therapeutic molecules Download PDFInfo
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- WO2001042200A1 WO2001042200A1 PCT/US2000/002657 US0002657W WO0142200A1 WO 2001042200 A1 WO2001042200 A1 WO 2001042200A1 US 0002657 W US0002657 W US 0002657W WO 0142200 A1 WO0142200 A1 WO 0142200A1
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- biologically active
- cationic
- amphiphile
- amphiphiles
- lipid
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- 0 CNC(OCCC(COC(NCCCNCCCCNCCCN)=O)OC(NC)=O)=* Chemical compound CNC(OCCC(COC(NCCCNCCCCNCCCN)=O)OC(NC)=O)=* 0.000 description 3
- ITROBPSSVYKXHA-UHFFFAOYSA-N NCCCNCCCCNCCCNC(OCC(CO)O)=O Chemical compound NCCCNCCCCNCCCNC(OCC(CO)O)=O ITROBPSSVYKXHA-UHFFFAOYSA-N 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
Definitions
- the present invention relates to novel cationic amphiphilic compounds that facilitate
- compositions that comprise such cationic
- amphiphiles and that are useful to deliver into the cells of patients therapeutically effective
- the invention are particularly useful in relation to gene therapy.
- the cell membrane presents a selectively-permeable barrier. Additionally, even when
- tissues is often not achieved include: (1) numerous proteins such as immunoglobins, (2)
- polynucleotides such as genomic DNA, cDNA, or mRNA (3) antisense polynucleotides
- 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 disorders
- dystrophy Representative of acquired disorders that can be treated are: (1) for cancers -
- Gene therapy 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 patterned thereon) into a cell.
- an expressible polynucleotide for example a gene, a cDNA, or an mRNA patterned thereon
- Cystic fibrosis a common lethal genetic disorder, is a particular example of a
- the disease that is a target for gene therapy.
- the disease is caused by the presence of one or
- cystic fibrosis transmembrane a protein known as cystic fibrosis transmembrane
- CTR conductance regulator
- active molecules must interact with both non-polar and polar environments (in or on, for
- the plasma membrane, tissue fluids, compartments within the cell, and the biologically active molecule itself are designed typically to contain both
- amphiphiles and many lipids and synthetic lipids that have been disclosed for use in
- amphiphiles One particularly important class of such amphiphiles is the cationic amphiphiles.
- cationic amphiphiles have polar groups that are capable of being positively
- amphiphiles interact with the many types of biologically
- active (therapeutic) molecules including, for example, negatively charged polynucleotides
- DOTMA dioleoyloxypropyll-N,N,N-trimethylammonium chloride
- DC-chol Additional compounds that facilitate transport of biologically active molecules
- ammonium bromide which is discussed below.
- the present invention provides for cationic amphiphiles that are particularly
- these cationic amphiphiles are particularly effective for intravenous delivery
- (E) is -(CH 2 )- or -NH- or is absent;
- R 1 is -NH-, an alkylamine, or a polyalkylamine
- R 2 is -NH-, an alkylamine, or a polyalkylamine and wherein R 1 is the same or is
- R 2 different from R 2 , except that both R' and R 2 cannot be NH;
- R 3 is H, or a saturated or unsaturated aliphatic group
- R 4 is H, or a saturated or unsaturated aliphatic group
- R 5 and R 6 are independently alkyl or acyl groups and may be saturated or contain
- amphiphiles may also be dimers of formula I which are provided
- (E) is -(CH 2 )- or -NH- or is absent;
- R 1 is -N-, an alkylamine, or a polyalkylamine
- R 2 is -N-, an alkylamine, or a polyalkylamine and wherein R 1 is the same or is
- R 1 and R 2 cannot be N;
- R 3 is H, or a saturated or unsaturated aliphatic group
- R 4 is H, or a saturated or unsaturated aliphatic group
- R 5 and R 6 are independently alkyl or acyl groups and may be saturated or contain
- compositions that comprise one or
- compositions facilitate intracellular delivery in the tissues of patients of therapeutically
- compositions of the invention are effective amounts of the biologically active molecules.
- the invention may be formulated to contain one or more additional physiologically
- the invention provides a method for facilitating the transfer of
- biologically active molecules into cells comprising the steps of: preparing a dispersion of a
- the cationic amphiphile(s) of the invention may be any suitable cationic amphiphile(s) of the invention.
- the cationic amphiphile(s) of the invention may be any suitable cationic amphiphile(s) of the invention.
- DOPE dioleoylphosphatidyl-ethanolamine
- compositions including those known as "DC-chol", and those disclosed by Behr et al. Proc. Natl. Acad.
- DOGS glycylspermine
- Yet another embodiment of the invention is improved expression and toxicity
- FIGURES 1 and 2 depict representative cationic amphiphiles.
- FIGURE 3 provides a map of the pCF2/CAT plasmid.
- This invention provides for cationic amphiphile compounds, and compositions
- amphiphiles are particularly useful in facilitating the transport of biologically
- DC-chol cholesterol
- DOGS dioctadecylamidolo-glycylspermine
- R' and R 2 as defined herein may have one or more positive charges in a
- Such cationic character may enhance
- amphiphile interaction of the amphiphile with therapeutic molecules (such as nucleic acids) or with cell
- nucleus or an endosomal vesicle thereof.
- the reader is referred to the
- amphiphiles none of which is to be taken as limiting on the practice of the present
- free base form or partially protonaled form are formulated with a co-lipid.
- (E) is -(CH 2 )- or -NH- or is absent;
- R 1 is -NH-, an alkylamine, or a polyalkylamine
- R 2 is -NH-, an alkylamine, or a polyalkylamine and wherein R' is the same or is
- R 3 is H, or a saturated or unsaturated aliphatic group
- R 4 is H, or a saturated or unsaturated aliphatic group; and R 5 and R 6 are independently alkyl or acyl groups and may be saturated or contain
- Cationic amphiphiles of the present invention may also be dimers of formula I which
- (E) is -(CH 2 )- or -NH- or is absent;
- R' is -N-, an alkylamine, or a polyalkylamine
- R 2 is -N-, an alkylamine, or a polyalkylamine and wherein R 1 is the same or is
- R 1 and R 2 cannot be N;
- R 3 is H, or a saturated or unsaturated aliphatic group
- R 4 is H, or a saturated or unsaturated aliphatic group; and R 5 and R 6 are independently alkyl or acyl groups and may be saturated or contain
- Preferred compounds representative of formula (I) include:
- practice of the invention include, for example, genomic DNA, cDNA, mRNA, antisense
- RNA or DNA RNA or DNA, polypeptides and small molecular weight drugs or hormones.
- compositions of the invention are provided.
- the biologically active molecule is an
- polynucleotide that is expressed when placed in the cells of a patient leading to the correction of a metabolic defect.
- the polynucleotide is expressed when placed in the cells of a patient leading to the correction of a metabolic defect.
- CFTR human cystic fibrosis transmembrane regulator
- R 5 and R 6 are independently alkyl or acyl groups, preferably containing about 8 to 30
- R' may be attached to R 2 through any carbon or nitrogen atom of R'
- R 3 and R 4 may be attached to R 1 and R 2 through and carbon or nitrogen atom
- polyalkylamines having a backbone length of about 40 nitrogen and carbon atoms, or more
- linking group (D) that connects the lipophilic group to the cationic
- linking group (D) is relatively short. It is generally preferred that within linking group (D) are
- inventions are those patterned on small amino acids such as glycinyl, alanyl, beta-alanyl,
- (D) is a linking group wherein no
- linking group (D) may be absent entirely.
- amphiphiles of the invention are active as
- mixing with one or more cationic amphiphiles include dioleoylphosphatidylethanolamine (“DOPE”), diphytanoylphosphatidylethanolamine, lyso-phosphatidylethanolamines other
- phosphatidylethanolamines phosphatidylcholines, lyso-phosphatidylcholines and
- a preferred molar ratio of cationic amphiphile to co-lipid is about
- co-lipids may react chemically with certain types of cationic amphiphiles under conditions
- the present invention may or may not contain such transacylation byproducts, or other
- compositions containing them Rather use of such compositions is within the practice of
- 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.
- compositions of the invention facilitate entry of biologically active molecules
- the compounds of the invention enables them to associate with the lipids of cell membranes
- liposome a vesicle
- liposomal compositions permit biologically active molecules carried therewith to gain
- amphiphiles of the invention need not form highly organized vesicles in order to be
- amphiphiles of the invention include: (a) polynucleotides such as
- genomic DNA cDNA, and mRNA that encode for therapeutically useful proteins as are
- RNA Ribonucleic acid
- antisense polynucleotides whether RNA or DNA
- Cationic amphiphile species of the invention may be blended so that two or more
- invention can also be blended for such use with amphiphiles that are known in the art.
- amphiphile(s) or of degradation products thereof the route of administration, the condition
- compositions of the invention are administered to the pharmaceutical compositions of the invention.
- Such preparations can be any preparations of the pharmaceutical compositions of the invention.
- Such preparations can be any preparations of the pharmaceutical compositions of the invention.
- Nebulizing devices, powder inhalers, and aerosolized solutions are representative of methods that may be used to administer such preparations to the
- compositions of the invention can in general be
- excipients such as the carbohydrates lactose, trehalose, sucrose, mannitol,
- maltose or galactose, and inorganic or organic salts may also be lyophilized (and then
- formulation for each amphiphile of the invention are capable of determination by those
- a principal aspect of the invention involves providing a composition
- a biologically active molecule for example, a polynucleotide
- cationic amphiphiles including optionally one or more co-lipids
- said resultant composition being in liquid or solid (preferably lyophilized) form, so that:
- amphiphiles of the invention in comparison with amphiphilic
- solute concentration may contribute positively to the successful transfection of
- composition provided in unbuffered water, contacts the target cells.
- compositions may therefore be incompatible with treating target tissues that
- formulations is subject to experimentation, but can be determined by those skilled in the art
- 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
- the cationic amphiphiles of the present invention preferably result from coupling a
- reaction was flushed with nitrogen, filtered and rinsed with ethanol to remove the
- 1,2-dilaurylaminocarboxylglycerol (0. 54g , 90%).
- compositions that comprise one or more of the cationic
- amphiphiles of the invention are amphiphiles of the invention.
- the biologically active portion of the amphiphiles of the invention are amphiphiles of the invention.
- the biologically active portion of the amphiphiles of the invention are amphiphiles of the invention.
- active macromolecule is an encoding DNA.
- FIG. 3 A map of pCFl /CAT is shown in Figure 3. Briefly, pCFl contains the enhancer/
- CMV cytomegalovirus
- intron is located between the promoter and the biologically active cDNA.
- polyadenylation signal of the bovine growth hormone gene was selected for placement
- the vector also contains a drug-resistance marker
- CTR transmembrane conductance regulator
- the pCFl vector is based on the commercially available vector pCMV ⁇ (Clontech).
- the pCMV ⁇ construct has a pUC 19 backbone (J. Vieira, et al., Gene 19: 259-268 (1982))
- 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
- bovine growth hormone polyadenylation signal sequence, the bovine growth hormone polyadenylation signal, pUC origin of replication and
- the pCFl -CFTR plasmid has been
- the human cytomegalovirus immediate early gene promoter and enhancer spans the
- nucleotides 1-639 This corresponds to the region from -522 to +72 relative to the transcriptional start site (+1) and includes almost the entire enhancer region from -524 to
- TATA box located at nucleotides 486-490 and the TATA box is at nucleotides 521-525 in pCFlCFTR.
- the CFTR transcript is predicted to initiate at nucleotide 548, which is the transcriptional
- the hybrid intron is composed of a fused tri-partite leader from adenovirus
- the elements in the intron are as follows: the first leader (nucleotides, 705-745), the second leader (nucleotides, 705-745), the second leader (nucleotides, 705-745), the second leader (nucleotides, 705-745), the second leader (nucleotides, 705-745), the second leader (nucleotides, 705-745), the second leader (nucleotides, 705-745), the second
- leader (nucleotides 746-816), the third leader (partial, nucleotides 817-877), the splice donor
- immunoglobulin gene splice donor sequence (nucleotides 1043-1 138).
- the donor site (G The donor site
- QT is at nucleotides 887-888
- G) is at nucleotides 1 128-1129 .
- the length of the intron is 230 nucleotides.
- the CFTR coding region comprises nucleotides
- the change at position 936 was introduced by site-directed mutagenesis and is silent but
- CFTR transcript comprises 51 nucleotides of the 3' untranslated region of the CFTR cDNA
- the BGH poly A signal contains 90 nucleotides of flanking sequence 5' to the
- the primary CFTR transcript is predicted to be cleaved downstream of the BGH
- amphiphiles of the present invention facilitate the intracellular delivery of biologically
- assay is a CAT assay, as described in U. S. Patent 5,840,710.
- Optimized compositions for in vivo testing are extrapolated from in vitro results.
- amphiphile concentration to DNA concentration is the amphiphile concentration to DNA concentration.
- Such a formulation can result in a ten-fold increase in transduction.
- Optimizing the 'neutral' co-lipid is also preferred.
- lipid to the neutral co-lipid is 1 :2 molar ratio. Also, substituting diphytanoylphosphatidyl
- mice in groups of four to five
- mice were injected with complex via their tail vein. The mice were then sacrificed 48 hours later and their lungs were harvested for assay of the CAT transgene. The absolute level of CAT
- amphiphile was "T" shaped.
- the anchor groups were dilauryl anchors, and cholesterol lipid
- anchor group having more than fourteen carbons.
- a thin film can be produced wherein the amphiphile and DOPE are
- DOPE ethanolamine
- amphiphile/co-lipid 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.
- amphiphile-containing film is then rehydrated in water for injection with gentle
- homogeneous phase for example, using a Puritan Bennett Raindrop nebulizer from Lenexa
- 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
- PEG-DMPE polyethylene glycol 5000 - dimyristoylphoshatidylethanolamine
- PEG-PE or other PEG derivatives may be used in replacement therefor.
- PEG derivatives such as PEG(5000)-DMPE is
- pCFl-CFTR plasmid (containing an encoding sequence for human cystic fibrosis
- transmembrane conductance regulator is provided in water-for-injection at a concentration
- DNA is delivered to the lung at a concentration thereof of between about 2 and about 12
- a sample of about 10 to about 40 ml is generally sufficient for one
- amphiphile/DNA will need to be repeated at time intervals of about two weeks, but
- cystic fibrosis For some disease states, such as cystic fibrosis, it is desirable to deliver biologically
- One method of intravenous delivery of the DNA complex can be performed as
- the reporter plasmid pCF-I CAT is purified to minimize endotoxin ( ⁇ 1
- amphiphile is provided as the free base.
- plasmid is prepared as a sodium salt in water, and the DOPE is provided in zwitterionic
- a female BALB/c mouse is injected intravenously using the tail vein.
- mice per group are used.
- the volume of Hpid:pDNA complex used is 100 ⁇ l.
- Organs can be frozen
- CAT chloramphenicol acetyl transferase
- amphiphiles There results the opportunity to substantially minimize side effects (such as
- amphiphile toxicity, inflammatory response that would otherwise affect adversely the
- compositions prepared together.
- An alternate method to produce such compositions may be as follows.
- the cationic amphiphile, the neutral co-lipid DOPE, and PEG(5000)DMPE are the neutral co-lipid DOPE, and PEG(5000)DMPE.
- 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
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Abstract
Novel cationic amphiphiles are provided that facilitate transport of biologically active (therapeutic) molecules into cells. There are provided also therapeutic compositions prepared typically bycontacting a dispersion of one or more cationic amphiphiles with the therapeutic molecules. Therapeutic molecules that can be delivered into cells according to the practice of the invention include DNA, RNA and polypeptides. Representative uses of the therapeutic compositions of the invention include providing gene therapy, and delivery of antisense polynucleotides or biologically active polypeptides to cells. With respect to therapeutic compositions for gene therapy, the DNA is provided typically in the form of a plasmid for complexing with the cationic amphiphile.
Description
Cationic Amphiphiles For Intracellular Delivery of Therapeutic Molecules
Background of the Invention
The present invention relates to novel cationic amphiphilic compounds that facilitate
the intracellular delivery of biologically active (therapeutic) molecules. The present
invention relates also to pharmaceutical compositions that comprise such cationic
amphiphiles, and that are useful to deliver into the cells of patients therapeutically effective
amounts of biologically active molecules. The novel cationic amphiphilic compounds of
the invention are particularly useful in relation to gene therapy.
Effective therapeutic use of many types of biologically active molecules has not
been achieved because methods are not available to cause delivery of therapeutically
effective amounts of such substances into the particular cells of a patient for which
treatment therewith would provide therapeutic benefit. Efficient delivery of therapeutically
sufficient amounts of such molecules into cells has often proved difficult since, for example,
the cell membrane presents a selectively-permeable barrier. Additionally, even when
biologically active molecules successfully enter targeted cells, they may be degraded
directly in the cell cytoplasm or even transported to structures in the cell, such as lyposomal
compartments, specialized for degradative processes. Thus, both the nature of substances
that are allowed to enter cells, and the amounts thereof that ultimately arrive at targeted
locations within cells, at which they can provide therapeutic benefit, are limited.
Although such selectivity is generally necessary in order that proper cell function
can be maintained, it comes with the disadvantage that many therapeutically valuable
substances (or therapeutically effective amounts thereof) are excluded. Additionally, the
complex structure, behavior, and environment presented by an intact tissue targeted for
intracellular delivery of biologically active molecules often interferes substantially with
such delivery, in comparison with the case presented by populations of cells cultured in
vitro.
Examples of biologically active molecules for which effective targeting to a patients'
tissues is often not achieved include: (1) numerous proteins such as immunoglobins, (2)
polynucleotides such as genomic DNA, cDNA, or mRNA (3) antisense polynucleotides; and
(4) many low molecular weight compounds, whether synthetic or naturally occurring, such
as the peptide hormones and antibiotics.
One of the fundamental challenges now facing the medical practitioner is that
although the defective genes that are associated with numerous inherited diseases (or that
represent disease risk factors including for various cancers) have been isolated and
characterized, methods to correct the disease states themselves by providing patients with
normal copies of such genes (the technique of gene therapy) are substantially lacking.
Accordingly, the development of improved methods of intracellular delivery therefor is of
great medical importance.
Examples of diseases that it is hoped can be treated by gene therapy include
inherited disorders such as cystic fibrosis, Gaucher's disease, Fabry's disease, and muscular
dystrophy. Representative of acquired disorders that can be treated are: (1) for cancers -
multiple myeloma, leukemias. melanomas, ovarian carcinoma and small cell lung cancer;
(2) for cardiovascular conditions - progressive heart failure, restenosis, and hemophilias;
and (3) for neurological conditions - traumatic brain injury.
Gene therapy 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 patterned thereon) into a cell.
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 (protein
replacement therapy) are often ineffective for the reasons mentioned above.
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. Abnormal
ion transport in airway cells leads to abnormal mucous secretion, inflammation and
infection, tissue damage, and eventually death.
It is widely hoped that gene therapy will provide a long lasting and predictable form
of therapy for certain disease states, and it is likely the only form of therapy suitable for
many inherited diseases. There remains however a critical need to develop compounds that
facilitate entry of functional genes into cells, and whose activity in this regard is sufficient
to provide for in vivo delivery of genes or other such biologically active therapeutic
molecules in concentrations thereof that are sufficient for intracellular therapeutic effect.
Reported Developments
In as much as compounds designed to facilitate intracellular delivery of biologically
active molecules must interact with both non-polar and polar environments ( in or on, for
example, the plasma membrane, tissue fluids, compartments within the cell, and the
biologically active molecule itself ), such compounds are designed typically to contain both
polar and non-polar domains. 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. One particularly important class of such amphiphiles is the cationic amphiphiles.
In general, cationic amphiphiles have polar groups that are capable of being positively
charged at or around physiological pH, and this property is understood in the art to be
important in defining how the amphiphiles interact with the many types of biologically
active (therapeutic) molecules including, for example, negatively charged polynucleotides
such as DNA.
Examples of 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. Feigner, et al., Proc. Natl. Acad. Sci. USA 84: 7413-
7417 (1987) disclose use of positively-charged synthetic cationic lipids including N-[l(2,3-
dioleoyloxy)propyll-N,N,N-trimethylammonium chloride ("DOTMA"), to form lipid/DNA
complexes suitable for transfections. See also Feigner et al., The Journal of Biological
Chemistry 269(4): 2550-2561 (1994). Behr et al., Proc. Natl. Acad. Sci. USA 86: 6982-
6986 (1989) disclose numerous amphiphiles including dioctadecylamidologlycylspermine
("DOGS"). U.S. Patent 5,283,185 to Epand et al. describes additional classes and species of
amphiphiles including 39 [N-(N1,N1 - dimethylaminoethane)carbamoyl] cholesterol, termed
"DC-chol." Additional compounds that facilitate transport of biologically active molecules
into cells are disclosed in U.S. Patent No. 5,264,618 to Feigner et al. See also Feigner et
al., The Journal Of Biological Chemistrv 269(4): 2550-2561 (1994) for disclosure therein of
further compounds including "DMRIE" 1,2- dimyristyloxypropyl-3-dimethyl-hydroxyethyl
ammonium bromide, which is discussed below. Reference to amphiphiles suitable for
intracellular delivery of biologically active molecules is also found in U.S. Patent No.
5,334,761 to Gebeyehu et al., and in Feigner et al., Methods in Enzvmology 5: 67-75
(1993).
Although the compounds mentioned in the above-identified references have been
demonstrated to facilitate (although in many such cases only in vitro) the entry of
biologically active molecules into cells, it is believed that the uptake efficiencies provided
thereby are insufficient to support numerous therapeutic applications, particularly gene
therapy. Additionally, since the above-identified compounds are understood to have only
modest activity, substantial quantities thereof must be used leading to concerns about the
toxicity of such compounds or of the metabolites thereof.
Moreover, several barriers to the systemic use of cationic lipid:pDNA complexes
have been identified. These include low, non-therapeutic levels of biologically active
molecule expression, aggregation and opsonization of the complexes and also abnormal
blood and liver parameters indicating acute toxicity.
Accordingly there is a need to develop a "second generation" of cationic amphiphiles
whose activity is so sufficient that successful therapies can be achieved therewith.
Summary of the Invention
The present invention provides for cationic amphiphiles that are particularly
effective to facilitate transport of biologically active molecules into cells. In a preferred
embodiment, these cationic amphiphiles are particularly effective for intravenous delivery
of biologically active molecules into cells. Representative amphiphiles are provided
according to the following structure:
wherein:
(A) and (B) are independently O, N, S or -O(C=0);
(C) is chosen from -CH2-, >C=O, and >C=S;
(D) is a linking group, which may be chosen from but is not limited to: -NH(C=O)-
-O(C=O)-; -(CH2)2-; -(CH2)3-; -(CH2)-(C=O)-; and -NH-(C=O)-O- or (D) is absent;
(E) is -(CH2)- or -NH- or is absent;
R1 is -NH-, an alkylamine, or a polyalkylamine;
R2 is -NH-, an alkylamine, or a polyalkylamine and wherein R1 is the same or is
different from R2, except that both R' and R2 cannot be NH;
R3 is H, or a saturated or unsaturated aliphatic group;
R4 is H, or a saturated or unsaturated aliphatic group; and
R5 and R6 are independently alkyl or acyl groups and may be saturated or contain
sites of unsaturation.
Representative amphiphiles may also be dimers of formula I which are provided
according to the following structure:
wherein:
(A) and (B) are independently O, N, S or -O(C=0);
(C) is chosen from -CH2-, >C=O, and >C=S;
(D) is a linking group, which may be chosen from but is not limited to: -NH(C=O)-;
-O(C=O)-; -(CH2)2-; -(CH2)3-; -(CH2)-(C=O)-; and -NH-(C=O)-O- or (D) is absent;
(E) is -(CH2)- or -NH- or is absent;
R1 is -N-, an alkylamine, or a polyalkylamine;
R2 is -N-, an alkylamine, or a polyalkylamine and wherein R1 is the same or is
different from R2, except that both R1 and R2 cannot be N;
R3 is H, or a saturated or unsaturated aliphatic group;
R4 is H, or a saturated or unsaturated aliphatic group; and
R5 and R6 are independently alkyl or acyl groups and may be saturated or contain
sites of unsaturation.
The invention provides also for pharmaceutical compositions that comprise one or
more cationic amphiphiles, and one or more biologically active molecules, wherein said
compositions facilitate intracellular delivery in the tissues of patients of therapeutically
effective amounts of the biologically active molecules. The pharmaceutical compositions of
the invention may be formulated to contain one or more additional physiologically
acceptable substances that stabilize the compositions for storage and/or contribute to the
successful intracellular delivery of the biologically active molecules.
In a further aspect, the invention provides a method for facilitating the transfer of
biologically active molecules into cells comprising the steps of: preparing a dispersion of a
cationic amphiphile of the invention; contacting said dispersion with a biologically active
molecule to form a complex between said amphiphile and said molecule; and contacting
cells with said complex thereby facilitating transfer of said biologically-active molecule into
the cells.
For pharmaceutical use, the cationic amphiphile(s) of the invention may be
formulated with one or more additional cationic amphiphiles including those known in the
art, or with neutral co-lipids such as dioleoylphosphatidyl-ethanolamine, ("DOPE"), to
facilitate delivery to cells of the biologically active molecules. Additionally, compositions
including those known as "DC-chol", and those disclosed by Behr et al. Proc. Natl. Acad.
Sci. USA 86: 6982-6986 (1989), a representative structure of which is dioctadecylamidolo-
glycylspermine ("DOGS"), may be included in the formulations of the present invention.
In a still further embodiment of the invention, particular organs or tissues may be
targeted for gene therapy by intravenous administration of amphiphile/biologically active
molecule complexes and by adjusting the ratio of amphiphile to DNA in such complexes,
and/or by adjusting the apparent charge or zeta potential thereof.
Yet another embodiment of the invention, is improved expression and toxicity
profiles by slow infusion of the amphiphile/biologically active molecule complexes rather
than by administering the complex as a bolus.
Cationic amphiphiles of the invention representative of formula (I) provided above
are as follows:
FIGURES 1 and 2 depict representative cationic amphiphiles.
FIGURE 3 provides a map of the pCF2/CAT plasmid.
Detailed Description of the Invention
This invention provides for cationic amphiphile compounds, and compositions
containing them, that are useful to facilitate transport of biologically active molecules into
cells. The amphiphiles are particularly useful in facilitating the transport of biologically
active polynucleotides into cells, and in particular to the cells of patients for the purpose of
gene therapy.
Cationic amphiphiles according to the practice of the invention possess several novel
features. These features may be seen in comparison with, for example, cationic amphiphile
structures such as those disclosed in U. S. Patent No. 5,283,185 to Epand et al., a
representative structure of which is 3β [N-(N',N' - dimethylaminoethane)-carbamoyl]
cholesterol, commonly known as "DC-chol", and to those disclosed by Behr et al. Proc.
Natl. Acad. Sci. USA 86: 6982- 6986 (1989), a representative structure of which is
dioctadecylamidolo-glycylspermine ("DOGS").
In connection with the practice of the present invention, it is noted that "cationic"
means that the R' and R2 as defined herein, may have one or more positive charges in a
solution that is at or near physiological pH. Such cationic character may enhance
interaction of the amphiphile with therapeutic molecules (such as nucleic acids) or with cell
structures (such as plasma membrane glycoproteins) thereby contributing to successful entry
of the therapeutic molecules into cells, or processing within subcompartments (such as the
nucleus or an endosomal vesicle) thereof. In this regard, the reader is referred to the
numerous theories in the art concerning transfection enhancing function of cationic
amphiphiles, none of which is to be taken as limiting on the practice of the present
invention. It should be noted that in vitro and in vivo efficacy has also been determined for
cationic amphiphiles of the invention when such amphiphiles (either in fully deprotonated
"free base" form or partially protonaled form) are formulated with a co-lipid.
Cationic amphiphiles of the present invention are provided according to the
following formula:
wherein:
(A) and (B) are independently O, N, S or -O(C=0);
(C) is chosen from -CH2-, >C=O, and >C=S;
(D) is a linking group, which may be chosen from but is not limited to: -NH(C=O)-
-O(C=O)-; -(CH2)2-; -(CH2)3-; -(CH2)-(C=O)-; and -NH-(C=O)-O- or (D) is absent;
(E) is -(CH2)- or -NH- or is absent;
R1 is -NH-, an alkylamine, or a polyalkylamine;
R2 is -NH-, an alkylamine, or a polyalkylamine and wherein R' is the same or is
different from R2, except that both R1 and R2 cannot be NH;
R3 is H, or a saturated or unsaturated aliphatic group;
R4 is H, or a saturated or unsaturated aliphatic group; and
R5 and R6 are independently alkyl or acyl groups and may be saturated or contain
sites of unsaturation.
Cationic amphiphiles of the present invention may also be dimers of formula I which
are provided according to formula II:
wherein:
(A) and (B) are independently O, N, S or -O(C=0);
(C) is chosen from -CH2-, >C=O, and >C=S;
(D) is a linking group, which may be chosen from but is not limited to: -NH(C=O)-
-O(C=O)-; -(CH2)2-; -(CH2)3-; -(CH2)-(C=O)-; and -NH-(C=O)-O- or (D) is absent;
(E) is -(CH2)- or -NH- or is absent;
R' is -N-, an alkylamine, or a polyalkylamine;
R2 is -N-, an alkylamine, or a polyalkylamine and wherein R1 is the same or is
different from R2, except that both R1 and R2 cannot be N;
R3 is H, or a saturated or unsaturated aliphatic group;
R4 is H, or a saturated or unsaturated aliphatic group; and
R5 and R6 are independently alkyl or acyl groups and may be saturated or contain
sites of unsaturation.
Preferred compounds representative of formula (I) include:
GL 194
Biological molecules for which transport into cells can be facilitated according to the
practice of the invention include, for example, genomic DNA, cDNA, mRNA, antisense
RNA or DNA, polypeptides and small molecular weight drugs or hormones. Representative
examples thereof are mentioned below in connection with the description of pharmaceutical
compositions of the invention.
In an important embodiment of the invention, the biologically active molecule is an
encoding polynucleotide that is expressed when placed in the cells of a patient leading to the
correction of a metabolic defect. In a particularly important example, the polynucleotide
encodes for a polypeptide having an amino acid sequence sufficiently duplicative of that of
human cystic fibrosis transmembrane regulator ("CFTR") to allow possession of the
biological property of epithelial cell anion channel regulation.
In connection with the design of cationic amphiphiles of the invention capable of
facilitating transport of biologically active molecules into cells, the following considerations
are of note.
Table 1
Table 1
For R1 and R2
1) -NH-
2) -NH-(CH2)
3) -NH-(CH2)
4) -NH-(CH2)
5) -NH-(CH2)
6) -NH-(CH2) NH-(CH2)(4)-
7) -NH-(CH2), NH-(CH2)(2)-
8) -NH-(CH2), NH-(CH2)(3)-
9) -NH-(CH2) NH-(CH2)(z)- 10) NH-(CH2)(x)-NH-(CH2)(y)-NH-(CH2)(z)- 11) -NH-(CH2)(w)-NH-(CH2)(x)-NH-(CH2)(y)-NH-(CH2)(z)- 12) -NH-(CH2)(V)-NH-(CH2)(W)-NH-(CH2)(X)-NH-(CH2)(V)-NH-(CH2)(Z)- 13) -[NH-(CH2)(w)]m-[NH-(CH2)(x)]n-[[CH3(CH2)(y)]N]-(CH2)(z)- 14) -[NH-(CH2)(x)]n-[[CH3(CH2)(y)]N]-(CH2)(z)-
15) -[NH-(CH2)(w)]m - [NH-(CH2)(x)]n -[[CH3(CH2)(y)]N]-(CH2)(z)-
16) - [[CH3(CH2)(X)][CH3(CH2)(V)]N]-(CH2)(Z) - 17) -NH-(CH2)(Z)-NH-
18) -NH-(CH,)(y)-NH-(CH2)(z)-NH-
19) -NH-(CH2)(y)-CH=CH-(CH2)(z)- 20) -[NH-(CH2)(w)]p-[[CH3(CH2)(x)]N]-(CH2)(y)-[NH-(CH2)(z)]q-
For R3 and R4
(1) H-
(2) CH3-
(3) CH3-(CH2)2-
(4) CH3-(CH2)4-
(5) CH3-(CH2)(Z)-
(6) CH3-[CH3-(CH2)(z)]CH-
(7) CH3-[CH3-(CH2),]CH- (8) CH3-[[CH3-(CH2)(y)][CH3-(CH2)(z)]]C-
(9) CH3-(CH2)(z)-CH=CH-CH2-
(10) CH3-[CH3-(CH,)(y)-CH=CH-(CH2)(z)]CH-
(l l) CH3-[[CH3-(CH2)(w)-CH=CH-(CH2)(x)][CH3-(CH2)(y)-CH=CH-(CH2)(z)]]CH- (12) CH3-[CH3-(CH2)(y)]CH-(CH2)(z) -
R5 and R6 are independently alkyl or acyl groups, preferably containing about 8 to 30
carbon atoms, and such groups may contain one or more points of unsaturation. For the
dimers of formula (II), R' may be attached to R2 through any carbon or nitrogen atom of R'
and R2, while R3 and R4 may be attached to R1 and R2 through and carbon or nitrogen atom
of R1 and R2.
It is noted that a polyalkylamine group that is very long may interfere, for example,
with the solubility of the resultant amphiphile, or interfere with its ability to stably interact
with the biologically active molecule selected for intracellular delivery. In this regard,
polyalkylamines having a backbone length of about 40 nitrogen and carbon atoms, or more,
may not be suitable for inclusion in amphiphiles. It is preferred that said backbone length
be about 30 nitrogen and carbon atoms in length, or less. However, for each such proposed
structure, its properties may be determined by experimentation, and its use is nonetheless
within the practice of the invention.
The Linking Group
Preferably the linking group (D) that connects the lipophilic group to the cationic
group is relatively short. It is generally preferred that within linking group (D) are
contained no more than about three or four atoms that themselves form a bridge of covalent
bonds between (E) and the lipophilic group.
Examples of (D) groups include -NH(C=O)-; -O(C=O)-; -(CH2)2-; -(CH2)3-;
-(CH2)-(C=O)-; and -NH-(C=O)-O-. Additional linking groups useful in the practice of the
invention are those patterned on small amino acids such as glycinyl, alanyl, beta-alanyl,
serinyl, threoninyl, and the like.
In certain preferred embodiments of the invention, (D) is a linking group wherein no
more than one atom of this group forms a bond with both (E) and the lipophilic group.
Alternatively, the linking group (D) may be absent entirely.
Co-lipids
It is generally believed in the art that preparing cationic amphiphiles as complexes
with co-lipids (particularly neutral co-lipids) enhances the capability of the amphiphile to
facilitate transfections. Although colipid-enhanced performance has been observed for
numerous of the amphiphiles of the invention, the amphiphiles of the invention are active as
transfectants without co-lipid. Accordingly, the practice of the present invention is neither
to be considered limited by theories as to co-lipid participation in intracellular delivery
mechanisms, nor to require the involvement of co-lipids.
Representative co-lipids that are useful according to the practice of the invention for
mixing with one or more cationic amphiphiles include dioleoylphosphatidylethanolamine
("DOPE"), diphytanoylphosphatidylethanolamine, lyso-phosphatidylethanolamines other
phosphatidylethanolamines, phosphatidylcholines, lyso-phosphatidylcholines and
cholesterol. Typically, a preferred molar ratio of cationic amphiphile to co-lipid is about
1 :1. However, it is within the practice of the invention to vary this ratio, including also over
a considerable range.
Transacylation Reactions
Although heretofore unrecognized in the art, it has been determined also that certain
co-lipids may react chemically with certain types of cationic amphiphiles under conditions
of co-storage, resulting in new molecular species. Generation of such new species is
believed to occur via mechanisms such as transacylation. For a further discussion thereof,
see international patent publication WO 96/18372 at pages 43-44, and also Figure 4 thereof.
It is to be understood that therapeutically effective pharmaceutical 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.
Preparation of Pharmaceutical Compositions and Administration Thereof
The present invention provides for pharmaceutical compositions that facilitate
intracellular delivery of therapeutically effective amounts of biologically active molecules.
Pharmaceutical 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 cell 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. By fusing with cell membranes,
such 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. However, unlike the case for many classes of amphiphiles or other lipid-like
molecules that have been proposed for use in therapeutic compositions, 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.
Biologically active molecules that can be provided intracellularly in therapeutic
amounts using the amphiphiles of the invention include: (a) polynucleotides such as
genomic DNA, cDNA, and mRNA that encode for therapeutically useful proteins as are
known in the art, (b) ribosomal RNA; (c) 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; and (d) ribozymes.
In general, and owing to the potential for leakage of contents therefrom, vesicles or
other structures formed from many 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 hormones and antibiotics.
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.
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, intravenously, 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.
Additionally, the therapeutic 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. Conditions of optimized
formulation for each amphiphile of the invention are capable of determination by those
skilled in the pharmaceutical art.
Accordingly, 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 solid (preferably lyophilized) form, so that: (1)
the therapeutic activity of the biologically active molecules is substantially preserved; (2)
the transfection-enhancing nature of the amphiphile (or of amphiphile/ DNA complex) is
maintained. Without being limited as to theory, it is believed that the excipients stabilize
the interaction of the amphiphile and biologically active molecule through one or more
effects including: (1) minimizing interactions with container surfaces, (2) preventing
irreversible aggregation of the complexes, and (3) maintaining amphiphile/DNA complexes
in a chemically-stable state, i.e., preventing oxidation and/or hydrolysis.
Although the presence of excipients in the pharmaceutical compositions of the
invention stabilizes the compositions and facilitates storage and manipulation thereof, it has
also been determined that moderate concentrations of numerous excipients may interfere
with the transfection enhancing capability of pharmaceutical formulations containing them.
In this regard, 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.
Selection of optimum concentrations of particular excipients for particular
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.
Another aspect of this invention concerns more effective gene transduction
following intravenous delivery of cationic lipid:pDNA complexes, using the novel series of
cationic lipids.
The invention will be further clarified by the following examples, which are
intended to be illustrative of the invention, but not limiting thereof.
-2.4- Examples
Example 1 - Methods of Synthesis
The cationic amphiphiles of the present invention preferably result from coupling a
polycationic headgroup to the lipophilic moiety through a primary amine. The following
methods illustrate production of certain of the cationic amphiphiles of the invention. Those
skilled in the art will recognize other methods to produce these compounds, and to produce
also other compounds of the invention.
Synthesis of 2,3-dilaurylglycerol N 4 Spermine Carbamate (Lipid 202)
3-OBn-glycerol (1. 00 g, 5. 49 mmol) in THF (20 mL) was added to a suspension of
sodium hydride (60% w/w in oil, 550 mg, 13. 725 mmol) in THF (30 mL) and allowed to
reflux overnight under dry nitrogen. A solution of dodecyl methane sulfonate (3. 39 g, 12.
078 mmol) in THF (20 mL) was added and the reaction was refluxed for another two days.
After cooling to room temperature, the reaction was filtered through a bed of Celite, rinsing
with THF. The filtrate was reduced in vacuo to a yellow oil which was redissolved in
diethyl ether (100 mL). The ether solution was washed with 0. 1 N NaOH (30 mL) and
dH2O (2 x 30 mL). The organic layer was dried over magnesium sulfate, filtered and
reduced in vacuo to a red-brown oil. The crude material was purified by flash column
chromatography (300 g silica gel) eluting with 3% ethyl acetate/hexanes. The desired
product was isolated as a pale yellow oil and characterized by IH NMR as 3-OBn-l,2-
dilaurylglycerol (1. 70 g, 60%).
3-OBn-l,2-dilaurylglycerol (1. 70 g, 3. 28 mmol) in ethanol (100 mL) was stirred
with 10% Pd/C (250 mg, 15 wt%) under a hydrogen atmosphere for 24 hours. The reaction
was flushed with nitrogen and filtered through Celite, rinsing with ethanol, to remove the
catalyst. The filtrate was reduced in vacuo to a solid. The crude material was purified by
flash column chromatography (140 g silica gel) eluting with 10% ethyl acetate/ hexanes.
The desired product was isolated as a white solid and characterized by 'H NMR as 1,2-
dilaurylglycerol (1. 23g, 88%).
4-Nitrophenylchloroformate (3. 02 g, 14. 98 mmol) was added to a chilled (0 °C) solution
of 1 ,2-dilaurylglycerol (4. 58 g, 10. 68 mmol) in methylene chloride (40 mL). The reaction
was stirred for 10 minutes at 0 °C and brought to room temperature and stirred for an
additional 30-45 minutes. Pyridine (1. 2 mL 14. 84 mmol) was added dropwise and the
reaction was allowed to stir. After 3 hours, the reaction was diluted with chloroform (150
mL). The chloroform solution was washed with saturated sodium bicarbonate (3 x 70 mL)
and saturated sodium chloride solution (100 mL). The organic layer was dried over
magnesium sulfate, filtered, and reduced in vacuo to a colorless oil. The colorless oil was
further dried under high vacuum for several hours and characterized by 'H NMR as the 4-
nitrophenylcarbonate of 1.2-dilaurylglycerol.
The carbonate was redissolved in a solution of DMF (40 mL ) and methylene
chloride (30 mL) and was added dropwise to a cooled (0 °C) solution of spermine (19. 42 g,
95. 97 mmol) and diisopropylethylamine ( 2 mL, 1 1. 48 mmol) in DMF (70 mL) and
methylene chloride (40 mL). The reaction was brought to room temperature and stirred for
2 hours. The DMF was removed in vacuo. The resultant oil was redissolved in 2:1
methylene chloride: methanol. A yellow precipitate was filtered out and washed several
times with additional 2:1. The filtrate was collected. Total volume of filtrate was
approximately 150 mL. This solution was washed with IM NaOH (3 x70 mL) and dH2O (2
x 70 mL). The organic layer was dried over sodium sulfate, filtered and reduced to an oil in
vacuo. The crude material was purified by flash column chromatography (500g silica gel)
eluting with 40:25:5, chloroform: methanol: ammonium hydroxide. The desired product
was isolated as a clear oil (4. 52 g, 60% ) and characterized by 'H NMR as Lipid 202, 2,3-
dilaurylglycerol N 4-spermine carbamate.
Synthesis of 3-(l '-sperminecarboxyl)-l ,2-dilauryIaminocarboxyIglycerol (Lipid
194)
3-OBn-glycerol (0. 25 gl. 37 mmol) and dodecyl isocyanate (1. 1 g, 4. 8 mmol) in
toluene (25 mL) was refluxed under Nitrogen for 18h. After cooling to room temperature
the solvent was removed in vacuo . The crude material was purified by flash column
chromatography (150 g silica gel) eluting with 20% ethyl acetate/ hexanes. The desired
product was isolated as a colorless oil and characterized by Η NMR as 3-OBn-l,2-
dilaurylaminocarboxylglycerol (0. 71 g, 86%).
3-OBn-l,2-dilaurylaminocarboxylglycerol (0. 71 g, 1. 17 mmol) in ethanol (35 mL)
was stirred with 10% Pd on Carbon (180 mg) under a hydrogen atmosphere for 48 hours.
The reaction was flushed with nitrogen, filtered and rinsed with ethanol to remove the
catalyst. The filtrate was reduced in vacuo to a gray solid. The crude material was
purified by flash column chromatography (70 g silica gel) eluting with 35% ethyl acetate/
hexanes. The desired product was isolated as a white solid and characterized by 'H NMR as
1,2-dilaurylaminocarboxylglycerol (0. 54g , 90%).
A solution of phosgene in toluene (1. 94M, 1. 6 mL) was added to the 1,2-
dilaurylaminocarboxylglycerol (0. 32 g, 0. 62 mmol) in methylene chloride (40 mL) and the
solution was stirred under nitrogen at room temperature for 18 h. A solution of imidazole
(0. 445 g, 6. 51 mmol) in methylene chloride (7. 5 mL) was added. After stirring for 2 h at
room temperature, methylene chloride (50 mL) was added and the organic layer was
washed with 15 % citric acid solution. The organic layer was dried with sodium sulfate and
the solvent removed in vacuo. The crude imidazole formate was dissolved in methylene
chloride (50 mL). Spermine (0. 63 g, 3. 1 mmol) and dimethylaminopyridine (10 mg, 0. 09
mmol) was added. The solution was stirred at room temperature for 18 h. Methylene
chloride (50 mL) was added and the organic layer was washed with water (25 mL). The
organic layer was dried with sodium sulfate and the solvent removed in vacuo. The crude
material was purified by flash column chromatography (45 g silica gel) eluting in succession
with 40:25 chloroform: methanol, 40:25:2 chloroform: methanol: cone, ammonium
hydroxide, 40:25:5 chloroform: methanol: cone, ammonium hydroxide, and finally
40:25:10 chloroform: methanol: cone, ammonium hydroxide. The desired product was
isolated and characterized by 'H NMR as Lipid 194, 3-(l'-sperminecarboxyl)-l,2-
dilaurylaminocarboxylglycerol (0. 26g, 56%) .
Example 2 - Construction of vectors
As aforementioned, numerous types of biologically active molecules can be
transported into cells in therapeutic compositions that comprise one or more of the cationic
amphiphiles of the invention. In an important embodiment of the invention, the biologically
active macromolecule is an encoding DNA. There follows a description of novel vectors
(plasmids) that are preferred in order to facilitate expression of such encoding DNAs in
target cells.
Construction of 12CF1
A map of pCFl /CAT is shown in Figure 3. Briefly, pCFl contains the enhancer/
promoter region from the immediate early gene of cytomegalovirus (CMV) . A hybrid
intron is located between the promoter and the biologically active cDNA. The
polyadenylation signal of the bovine growth hormone gene was selected for placement
downstream from the biologically active. The vector also contains a drug-resistance marker
that encodes the aminoglycosidase X-phosphotransferase gene (derived from the transposon
Tn903, A. Oka et al., Journal of Molecular Biology 147: 217-226 (1981)) thereby conferring
resistance to kanamycin. Further details of pCFl structure are provided directly below,
including description of placement therein of a cDNA sequence encoding for cystic fibrosis
transmembrane conductance regulator (CFTR) protein.
The pCFl vector is based on the commercially available vector pCMVβ (Clontech).
The pCMVβ construct has a pUC 19 backbone (J. Vieira, et al., Gene 19: 259-268 (1982))
that includes a prokaryotic origin of replication derived originally from pBR322.
Basic features of the pCFl -plasmid (as constructed to include a nucleotide sequence
coding for CFTR) are as follows. Proceeding clockwise, the basic features are: 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 pCFl -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 to +72 relative to
the transcriptional start site (+1) and includes almost the entire enhancer region from -524 to
-118 as originally defined by Boshart et al., Cell 41 : 521-530 (1985). The CAAT box is
located at nucleotides 486-490 and the TATA box is at nucleotides 521-525 in pCFlCFTR.
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 sequence (nucleotides 1043-1 138). The donor site (G |
QT is at nucleotides 887-888, the acceptor site (AG | G) is at nucleotides 1 128-1129 , and
the length of the intron is 230 nucleotides. The CFTR coding region comprises nucleotides
1 183-5622.
Within the CFTR-encoding cDNA of pCFI-CFTR, there are two differences from
the originally-published predicted cDNA sequence (J. Riordan et al., Science 245: 1066-
1073 (1989)); (1) an A to C change at position 1990 of the CFTR cDNA which corrects an
error in the original published sequence, and (2) a T to C change introduced at position 936.
The change at position 936 was introduced by site-directed mutagenesis and is silent but
greatly increases the stability of the cDNA when propagated in bacterial plasmids. See R. J.
Gregory et al., Nature 347: 382-386 (1990). The 3' untranslated region of the predicted
CFTR transcript comprises 51 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. There is a deletion in pCFl-CFTR at position
+46 relative to the cleavage site, but the deletion is not predicted to effect either
polyadenylation efficiency or cleavage site accuracy, based on the studies of E.C. Goodwin
et al., J. Biol. Chem. 267: 16330-16334 (1992). After the addition of a poly A tail, the size
of the resulting transcript is approximately 5.1 kb.
Example 3 - Methods of Using A Pharmaceutical Composition According to the
Invention
Cell Transfection
Cell transfection assays are well known in the art. Examples of such assays are
present in U. S. Patent No. 5,840,710, herein incorporated by reference. The cationic
amphiphiles of the present invention facilitate the intracellular delivery of biologically
active molecules by the use of such cell transfection assays.
CAT Assay
Assays to assess the ability of the cationic amphiphiles of the invention to transfect
cells in vivo from live specimens are also well known in the art. One example of such an
assay is a CAT assay, as described in U. S. Patent 5,840,710.
Optimized compositions for in vivo testing are extrapolated from in vitro results.
This facilitates the screening of large numbers of amphiphiles and produces broadly, if not
precisely, comparable data. Thus, the ratio, for in vivo testing, of amphiphile concentration
to DOPE concentration, is taken from the in vitro experiments, as is the optimized ratio of
amphiphile concentration to DNA concentration.
Choosing a Cationic Amphiphile Formulation
In developing a non-viral gene therapy vector for treating a disease state, the
following set of rules governing the systemic delivery of cationic lipid:pDNA complexes for
optimal expression should be considered to provide effective gene transduction following
intravenous delivery of cationic lipid:pDNA complexes. It is preferred to couple the
polycationic headgroup to the lipophilic moiety through a primary amine group rather than
through a secondary amine. Such a coupling results in a linear molecule rather than a "T"
shaped molecule, and can result in a ten-fold increase in expression.
It is also preferred to formulate the cationic amines as free bases rather than
protonated. Such a formulation can result in a ten-fold increase in transduction.
Optimizing the 'neutral' co-lipid is also preferred. A preferred molar ratio of cationic
lipid to the neutral co-lipid is 1 :2 molar ratio. Also, substituting diphytanoylphosphatidyl
ethanolamine for the commonly used DOPE results in a significant increase in expression.
In fact, including a low (0.05) mole percentage DMPE-PEGjooo in the lipid formulation has
been found to inhibit aggregation of the cationic lipid:pDNA complexes at the high
concentrations required for the most effective transduction.
In view of the foregoing, the following experiments were performed. A complex
was prepared by mixing equal volumes of the plasmid DNA and the cationic amphiphile of
the invention, both in water, and leaving them to complex for 15 minutes. The complex was
lOOμl of cationic lipid:pDNA at a ratio of 1 :lmM. BALB/c mice in groups of four to five
were injected with complex via their tail vein. The mice were then sacrificed 48 hours later
and their lungs were harvested for assay of the CAT transgene. The absolute level of CAT
is determined and reported or presented as a proportion of a control group where
appropriate.
Comparative experiments were performed as described above except the cationic
amphiphile was "T" shaped. In other words, a series of matched lipids, differing only in the
orientation of the headgroup, were tested, i.e., the lipid anchor groups in these matched
lipids were identical. The anchor groups were dilauryl anchors, and cholesterol lipid
anchors.
These experiments demonstrated a good correlation between the orientation of
polycationic headgroups and transfection efficiency in the lung. Those cationic amphiphiles
that were coupled through a secondary amine, i.e., forming a "T"-shape, were generally
more effective intranasally than if coupled through a primary amine, i.e., forming a linear
shape. However, the linear shape yielded a higher level of expression in intravenous
delivery.
In addition, these experiments demonstrated that an alkyl lipid anchor provides
improved delivery as compared to a cholesterol lipid anchor. Moreover, a C,2 alkyl anchor
provides improved delivery as compared to a Cuι, C,4 alkyl lipid anchor, or an alkyl lipid
anchor group having more than fourteen carbons.
Formulating the Pharmaceutical Compositions
A recommended procedure for formulating the pharmaceutical compositions of the
invention is as follows. A thin film can be produced wherein the amphiphile and DOPE are
present in a molar ratio of 1 : 1 , generally as follows.
Separate samples of an amphiphile and the neutral lipid dioleoylphosphatidyl
ethanolamine ("DOPE") are dissolved in chloroform as stock preparations. Following
combination of the solutions (as a 1 : 1 molar composition), a thin film is 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 amphiphile-containing film is then rehydrated in water for injection with gentle
vortexing to a resultant amphiphile concentration of about 3mM. However, in order to
increase the amount of amphiphile/DNA complex that may be stably delivered as a
homogeneous phase (for example, using a Puritan Bennett Raindrop nebulizer from Lenexa
Medical Division, Lenexa, KS, or the PARI LC JetTM nebulizer from PARI Respiratory
Equipment, Inc., Richmond, VA), 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. [A suitable source of
PEG-DMPE, polyethylene glycol 5000 - dimyristoylphoshatidylethanolamine, is Catalog
No. 880210 from Avanti Polar Lipids, Alabaster, AL]. Additional fatty acid species of
PEG-PE or other PEG derivatives may be used in replacement therefor.
Without being limited as to theory, PEG derivatives, such as 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.
pCFl-CFTR plasmid (containing an encoding sequence for human cystic fibrosis
transmembrane conductance regulator) 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 two solutions for a period of 10 minutes.
Using the Pharmaceutical Compositions
One method of delivering the DNA to the lung is by aerosolization. The aerosolized
DNA is delivered 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 A F508
mutation in the CFTR-encoding gene.
It is expected that this procedure (using a freshly prepared sample of
amphiphile/DNA) will need to be repeated at time intervals of about two weeks, but
depending considerably upon the response of the patient, duration of expression from the
transfected DNA, and the appearance of any potential adverse effects such as inflammation,
all of which can be determined for each individual patient and taken into account by the
patient's physicians.
It is presently preferred to deliver solutions intravenously. Moreover, it is preferred
to deliver the amphiphile/biologically active molecule complex by slow infusion. Such
slow infusion improves expression and toxicity profiles as compared to delivery as a bolus.
For some disease states, such as cystic fibrosis, it is desirable to deliver biologically
active molecules to the lung. While delivery by aerosol is the most direct approach to
achieve this goal, there are difficulties inherent with such delivery. Moreover, with the
potential need to target organs other than the lung (for example, the pancreas for cystic
fibrosis), it is important to evaluate the feasibility of lung delivery using non-aerosol
delivery formats.
One method of intravenous delivery of the DNA complex can be performed as
follows. The reporter plasmid pCF-I CAT is purified to minimize endotoxin (<1
EU/mg pDNA), and also chromosomal DNA contamination (< 2%). An amphiphile
according to the invention (1 :1 with DOPE) / DNA complex was prepared according to the
procedures set forth above. Preferably, the amphiphile is provided as the free base. The
plasmid is prepared as a sodium salt in water, and the DOPE is provided in zwitterionic
form.
A female BALB/c mouse is injected intravenously using the tail vein. Preferably 5
animals per group are used. The volume of Hpid:pDNA complex used is 100 μl. The mice
are sacrificed 48 h following administration of the complex. Organs can be frozen
immediately on dry ice to store for subsequent analysis.
Expression of chloramphenicol acetyl transferase (CAT) is quantitated using a
radiochemical assay for CAT enzymatic activity. Organs are weighed and homogenized on
ice in a lysis buffer containing protease inhibitors. The lysate is freeze-thawed 3X,
centrifuged, and heated to 65°C to inactivate deacetylases before adding it to a reaction
mixture containing C]4-chloramphenicol. After an incubation at 37°C, the mixture is
extracted with ethyl acetate, concentrated, spotted onto TLC plates and eluted with
CHC13/MeOH. Spots corresponding to the acylated reaction products are then quantitated
(Betagen) and converted to ng CAT activity using authentic CAT standards.
One important advantage of the cationic amphiphiles of the present invention is that
they 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 otherwise affect adversely the
success of the gene therapy.
A further particular advantage associated with use of many of the amphiphiles of the
invention should again be mentioned. Many of the amphiphiles of the invention were
designed so that the metabolism thereof would rapidly proceed toward relatively harmless
biologically-compatible components.
Example 4 - Alternate Procedure to Prepare an Amphiphile/Co-lipid Composition
In order to formulate material that is suitable for clinical administration, it may be
preferable to avoid use of chloroform when the cationic amphiphile and the co-lipid are
prepared together. An alternate method to produce such compositions may be as follows.
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 until use.
The specification is most thoroughly understood in light of the teachings of the references cited within the specification which are hereby incorporated by reference. The embodiments within the specification provide an illustration of embodiments of the
invention and should not be construed to limit the scope of the invention. The skilled
artisan readily recognizes that many other embodiments are encompassed by the invention.
Claims
1. A cationic amphiphile effective for facilitating transport of at least one
biologically active molecule into a cell, said cationic amphiphile having the structure:
wherein:
(A) and (B) are independently O, N, S or -O(C=0);
(C) is -CH2-, >C=O, or >C=S;
(D) is a linking group or is absent;
(E) is -(CH2)- or -NH- or is absent;
R1 is -NH-, an alkylamine, or a polyalkylamine;
R2 is -NH-, an alkylamine, or a polyalkylamine and wherein said R1 is the same or is
different from said R2, except that both said R1 and R2 cannot be NH;
R3 is H, or a saturated or unsaturated aliphatic group;
R4 is H, or a saturated or unsaturated aliphatic group; and R5 and R6 are independently alkyl or acyl groups and may be saturated or contain
sites of unsaturation.
2. A cationic amphiphile according to claim 1 , wherein said linking group, (D),
is -NH(C=O)-; -O(C=O)-; -(CH2)2-; -(CH2)3-; -(CH2)-(C=O)-; or -NH-(C=O)-O-.
3. A composition effective for facilitating transport of at least one biologically
active molecule into a cell, said composition comprising:
said cationic amphiphile according to claim 1 ; and
a biologically active molecule.
4. A composition according to claim 3, further comprising a co-lipid.
5. A composition according to claim 4, wherein said co-lipid is
diphytanoylphosphatidylethanolamine.
6. A composition according to claim 3, further comprising a PEG derivative.
7. A cationic amphiphile according to claim 1, wherein said cationic
amphiphile is
GL 194
8. A cationic amphip ile according to claim 1, wherein said cationic
amphiphile is
9. A cationic amphiphile effective for facilitating transport of at least one
biologically active molecule into a cell, said cationic amphiphile having the structure:
wherein:
(A) and (B) are independently O, N, S or -O(C=0);
(C) is -CH2-, >C=O, or >C=S; (D) is a linking group or is absent;
(E) is -(CH2)- or -NH- or is absent;
R1 is -N-, an alkylamine, or a polyalkylamine;
R2 is -N-, an alkylamine, or a polyalkylamine and wherein said R1 is the same or is
different from said R2, except that both said R1 and R2 cannot be N;
R3 is H, or a saturated or unsaturated aliphatic group;
R4 is H, or a saturated or unsaturated aliphatic group; and
R5 and R6 are independently alkyl or acyl groups and may be saturated or contain
sites of unsaturation.
10. A cationic amphiphile according to claim 9, wherein said linking group (D)
is -NH(C=O)-; -O(C=O)-; -(CH2)2-; -(CH2)3-; -(CH2)-(C=O)-; or -NH-(C=O)-O-.
11. A composition effective for facilitating transport of at least one biologically
active molecule into a cell, said composition comprising:
said cationic amphiphile according to claim 9; and
a biologically active molecule.
12. A composition according to claim 11 , further comprising a co-lipid.
13. A composition according to claim 12, wherein said co-lipid is
diphytanoylphosphatidylethanolamine.
14. A composition according to claim 11 , further comprising a PEG derivative.
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