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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to lipophilic cationic compounds and several of
their uses. The invention also relates to a novel DNA transfection method,
in which the compounds of this invention can be used.
2. Related Art
Liposomes are microscopic vesicles consisting of concentric lipid bilayers.
Structurally, liposomes range in size and shape from long tubes to
spheres, with dimensions from a few hundred Angstroms to fractions of a
millimeter. Regardless of the overall shape, the bilayers are generally
organized as closed concentric lamellae, with an aqueous layer separating
each lamella from its neighbor. Vesicle size normally falls in a range of
between about 20 and about 30,000 nm in diameter. The liquid film between
lamellae is usually between about 3 and 10 nm.
Typically, liposomes can be divided into three categories based on their
overall size and the nature of the lamellar structure. The three
classifications, as developed by the New York Academy Sciences Meeting,
"Liposomes and Their Use in Biology and Medicine," of December 1977, are
multi-lamellar vesicles (MLV's), small uni-lamellar vesicles (SUV's) and
large uni-lamellar vesicles (LUV's).
SUV's range in diameter from approximately 20 to 50 nm and consist of a
single lipid bilayer surrounding an aqueous compartment. Unilamellar
vesicles can also be prepared in sizes from about 50 nm to 600 nm in
diameter. While unilamellar are single compartmental vesicles of fairly
uniform size, MLV's vary greatly in size up to 10,000 nm, or thereabouts,
are multi-compartmental in their structure and contain more than one
bilayer. LUV liposomes are so named because of their large diameter which
ranges from about 600 nm to 30,000 nm; they can contain more than one
bilayer.
Liposomes may be prepared by a number of methods not all of which produce
the three different types of liposomes. For example, ultrasonic dispersion
by means of immersing a metal probe directly into a suspension of MLV's is
a common way for preparing SUV's.
Preparing liposomes of the MLV class usually involves dissolving the lipids
in an appropriate organic solvent and then removing the solvent under a
gas or air stream. This leaves behind a thin film of dry lipid on the
surface of the container. An aqueous solution is then introduced into the
container with shaking in order to free lipid material from the sides of
the container. This process disperses the lipid, causing it to form into
lipid aggregates or liposomes.
Liposomes of the LUV variety may be made by slow hydration of a thin layer
of lipid with distilled water or an aqueous solution of some sort.
Alternatively, liposomes may be prepared by lyophilization. This process
comprises drying a solution of lipids to a film under a stream of
nitrogen. This film is then dissolved in a volatile solvent, frozen, and
placed on a lyophilization apparatus to remove the solvent. To prepare a
pharmaceutical formulation containing a drug, a solution of the drug is
added to the lyophilized lipids, whereupon liposomes are formed.
A variety of methods for preparing various liposome forms have been
described in the periodical and patent literature. For specific reviews
and information on liposome formulations, reference is made to reviews by
Pagano and Weinstein (Ann. Rev. Biophysic. Bioeng., 7, 435-68 (1978)) and
Szoka and Papahadjopoulos (Ann. Rev. Biophysic. Bioeng., 9, 467-508
(1980)) and additionally to a number of patents. for example, U.S. Pat.
Nos. 4,229,360; 4,224,179; 4,241,046; 4,078,052; and 4,235,871.
Thus, in the broadest terms, liposomes are prepared from one or more
lipids. Though it has been thought that any type of lipid could be used in
liposomes, e.g. cationic, neutral or anionic lipids, experience with
positively charged liposomes has indicated several problems which have not
been fully addressed to date. The amines which have to date been employed
in preparing cationic liposomes have either not been sufficiently
chemically stable to allow for the storage of the vesicle itself (short
shelf life) or the structure of the amines has been such that they can be
leached out of the liposome bilayer. One such amine, stearlylamine, has
toxicity concerns which limit its use as a component of liposomes in a
pharmaceutical formulation. Another amine, dimethyl dioctadecyl ammonium
bromide, lacks the appropriate molecular geometry for optimum formation of
the bilayers that comprise the liposome structure.
Various biological substances have been encapsulated into liposomes by
contacting a lipid with the matter to be encapsulated and then forming the
liposomes as described above. A drawback of this methodology, commonly
acknowledged by those familiar with the art, is that the fraction of
material encapsulated into the liposome structure is generally less than
50%, usually less than 20%, often necessitating an extra step to remove
unencapsulated material. An additional problem, related to the above, is
that after removal of unencapsulated material, the encapsulated material
can leak out of the liposome. This second issue represents a substantial
stability problem to which much attention has been addressed in the art.
Liposomes have been used to introduce DNA into cells. More specifically,
various DNA transfection methodologies have been used, including
microinjection, protoplast fusion, liposome fusion, calcium phosphate
precipitation, electroporation and retroviruses. All of these methods
suffer from some significant drawbacks: they tend to be too inefficient,
too toxic, too complicated or too tedious to be conveniently and
effectively adapted to biological and/or therapeutic protocols on a large
scale. For instance, the calcium phosphate precipitation method can
successfully transfect only about 1 in 10.sup.7 to 1 in 10.sup.4 cells;
this frequency is too low to be applied to current biological and/or
therapeutic protocols. Microinjection is efficient but not practical for
large numbers of cells or for large numbers of patients. Protoplast fusion
is more efficient than the calcium phosphate method but the propylene
glycol that is required is toxic to the cells. Electroporation is more
efficient then calcium phosphate but requires a special apparatus.
Retroviruses are sufficiently efficient but the introduction of viruses
into the patient leads to concerns about infection and cancer. Liposomes
have been used before but the published protocols have not been shown to
be any more efficient than calcium phosphate. The most desirable
transfection method would involve one that gives very high efficiency
without the introduction of any toxic or infectious substances and be
simple to perform without a sophisticated apparatus. The method that we
describe satisfies all of these criteria.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, the compounds of this
invention are illustrated by Formula (I):
##STR2##
or an optical isomer thereof, wherein R.sup.1 and R.sup.2 are
independently an alkyl, alkenyl, or alkynyl group of 6 to 24 carbon atoms;
R.sup.3, R.sup.4 and R.sup.5 are independently hydrogen, alkyl of 1 to 8
carbon atoms, aryl or aralkyl of 6 to 11 carbon atoms; alternatively two
or three of R.sup.3, R.sup.4 and R.sup.5 are combined with the positively
charged nitrogen atom to form a cyclic structure having from 5 to 8 atoms,
where, in addition to the positively charged nitrogen atom, the atoms in
the structure are carbon atoms and can include one oxygen, nitrogen or
sulfur atom; n is 1 to 8; and X is an anion.
According to other aspects of the invention, liposome and pharmaceutical
formulations are claimed: said liposome formulations comprising up to 10%
by weight of a biologically active substance, 1% to 20% by weight of a
lipid component comprising a compound of Formula I in a quantity of from
about 1% to 100% by weight, and an aqueous solution in a quantity
sufficient to make 100% by volume; and said pharmaceutical formulations
comprising a therapeutically effective amount of a drug, an optional
pharmaceutically acceptable excipient, and a lipid component comprising a
compound of Formula I in a quantity of from about 1% to 100% by weight.
According to another aspect of the invention, a polyanion-lipid complex,
formed from a compound of Formula I and a polyanion, is claimed.
According to yet another aspect of the invention, a method is claimed for
forming a polyanion-lipid complex, said method comprising the steps of
contacting a liposomal composition prepared from a positively charged
liposome-forming lipid with a negatively charged polyanion.
According to still another aspect of the invention, a positively-charged
polynucelotide-liposome complex is claimed, comprising a lipid of Formula
I and a polynucleotide.
According to a further aspect of the invention, a method is claimed for
preparing a positively-charged polynucleotide-lipid complex. The method
comprises the steps of contacting a positively charged liposome made from
a lipid of Formula I with a polyanion.
According to yet another aspect of the invention, a method is claimed for
introducing a polyanion into a cell. The method comprises forming a
liposome from a lipid of Formula I, contacting the liposome with a
polyanion to form a positively-charged polyanion-liposome complex, and
incubating the complex with a cell.
According to still another aspect of the invention, a method is claimed for
intracellularly delivering a biologically active substance, which method
comprises forming a liposome comprising a lipid of Formula I and a
biologically active substance, and incubating the liposome with a cell
culture.
According to a further aspect of the invention, an antigenic formulation is
claimed, comprising an antigen and a compound of Formula I.
According to a still further aspect of the invention, a method is claimed
for the transdermal, topical or ocular delivery of a drug. The method
comprises the steps of forming a liposome comprising a compound of Formula
I and the drug; and applying the liposome to the skin or mucous membranes
of a human or animal subject.
According to another aspect of the invention, double coated liposome
complexes are claimed, comprising a polyanion, a lipid of Formula I, and a
negatively charged co-lipid.
According to a still further aspect of the invention, a method is claimed
for making said double-coated complexes, comprising forming a liposome
from a lipid of Formula I; contacting it with a polyanion; and contacting
the resulting complex with an excess of negatively-charged lipid.
DETAILED DESCRIPTION OF THE INVENTION
Several advantages flow from the compounds and methods of the present
invention. One of the advantages of the methods and materials disclosed
herein is that they permit up to 100% entrapment of polyanionic substances
by an exceedingly convenient and practical protocol. Another advantage of
the liposome compositions disclosed herein is that they are not subject to
instability due to leakage of the entrapped polyanionic substance. Still
another advantage is that the convenient and practical methodology
disclosed herein yields compositions of matter with unique properties
enabling entry of the entrapped polyanionic substance, such as DNA, into
living cells. This property of the resulting lipid/polyanion complex
enables the expression of biological activities to extends not previously
seen in these cells. And still further, this methodology leads to results
that have not been obtained with conventional liposomes.
The positively charged pharmaceutical formulations, particularly liposomes,
of this invention are pharmaceutically advantageous: the presentation of
positively charged materials to the negatively charged cell surface
results in better uptake of the pharmaceutical materials by he cells.
The unique advantages of the technology disclosed herein are of two types.
First, the compounds of Formula I represent novel positively charged
liposome forming lipids, which can be used for the formation of positively
charged liposomes in which drugs or other materials can be encapsulated in
the conventional manner. The uniqueness of this aspect of the invention
depends on the chemical structure of the compounds of Formula I. The
principal advantages of this structure derive from the geometry of the two
parallel aliphatic chains, the overall positive charge of the molecule
itself, and the chemical stability of the ether linkages. The geometry of
the two aliphatic chains enables the organization by the compounds of
Formula I into stable bilayer structures. These bilayers comprise the
overall structure of the liposome itself. The positive charge on the
molecules of Formula I provides the resulting liposome with an overall
positive charge, resulting in a net positively charged liposome. The ether
linkage of the aliphatic chains provides the chemical stability important
for the type of chemical structure synthesized and for the type of
applications described herein. Both hydrophobic and hydrophilic
biologically active substances can be incorporated into the resulting
liposomes using conventional liposome technology commonly known by those
familiar with the art. The resulting liposomes produced are better than
those produced with other commonly available materials, because the
compounds of Formula I have a geometry more compatible with the formation
of bilayers, leading to a liposome with greater physical stability.
Thus, compounds of Formula I do not suffer from the drawbacks of amines
employed in liposomes before this invention. The ether linkage of the
compounds of Formula I is highly stable in liposomes. Additionally, they
otherwise migrate out of the liposome matrix as do stearyl amines and
other amines. Moreover, concerns of toxicity are significantly reduced
with the compounds of Formula I. Still further, the parallel geometry of
the aliphatic chains in the preferred embodiments of the compounds of
Formula I overcomes problems with bilayer compatibility that are common to
molecules such as dioctadecyldimethyl ammonium bromide.
The second unique advantage of the technology disclosed herein is derived
from the novel method for incorporating polyanionic biologically active
substances into a liposome complex. This complex is composed of positively
charged liposomes prepared from compounds of Formula I or other positively
charged lipids, and a polyanionic substance. According to the method,
premade liposomes are contacted with the polyanionic substance in an
aqueous environment. The precise nature of the complex formed is
determined by the chemical composition of the positively charged liposomes
used and by the molar ratio of total positive charges on the liposome, to
the total negative charges on the polyanion. Precise tuning of these
compositional aspects determines the biological activity of the final
product produced. The advantages of this methodology over other liposome
technology commonly known in the art are that the new method results in up
to 100% entrapment of the biologically active substance, the entrapped
material does not leak out in storage, and the complex has unique
biological properties not shared by liposome encapsulated material
prepared in the conventional manner. Furthermore, by utilizing
double-coated complexes, preferential delivery to a specific site in the
body can be obtained in vivo, to ultimately provide site-specific
intracellular delivery via the positively-charged lipid complex portion of
the double-coated complex.
A. Definitions
An aliphatic chain comprises the classes of alkyl, alkenyl and alkynyl
defined below. A straight aliphatic chain is limited to unbranched carbon
chain radicals.
Alkyl refers to a fully saturated branched or unbranched carbon chain
radical having the number of carbon atoms specified, or up to 22 carbon
atoms if no specification is made. For example, alkyl of 1 to 8 carbon
atoms refers to radicals such as methyl, ethyl, propyl, butyl, pentyl,
hexyl, heptyl, and octyl, and those radicals which are positional isomers
of these radicals. Lower alkyl refers to alkyl of 1 to 4 carbon atoms,
such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secbutyl,
and tert-butyl. Alkyl of 6 to 24 carbon atoms includes hexyl, heptyl,
octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexandecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl,
docosyl, tricosyl and tetracosyl.
Alkenyl refers to any branched or unbranched unsaturated carbon chain
radical having the number of carbon atoms specified, or up to 22 carbon
atoms if no limitation on the number of carbon atoms is specified; and
having 1 or more double bonds in the radical. Alkenyl of 6 to 24 carbon
atoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl,
undecenyl, dodenyl, tridecenyl, tetradecenyul, pentadecenyl, hexadecenyl,
heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl,
docosenyl, tricosenyl and tetracosenyl, in their various isomeric forms,
where the unsaturated bond(s) can be located anywhere in the radical.
Alkynyl refers to hydrocarbon radicals of the scope of alkenyl, but having
1 or more triple bonds in the radical.
An antigen is any substance to which an organism can elicit an immune
response.
Antisense refers to a nucleotide sequence that is complementary to a
specific sequence of nucleotides in DNA or RNA.
Aryl refers to phenyl or naphthyl.
Aralkyl of 7 to 11 carbon atoms refers to a radical having an alkyl group
to which is attached a benzene ring such as the benzyl radical, phenethyl,
3-phenylpropyl, or the like.
Biologically active substance refers to any molecule or mixture or complex
of molecules that exerts a biological effect in vitro and/or in vivo,
including pharmaceuticals, drugs, proteins, vitamins, steroids,
polyanions, nucleosides, nucleotides, polynucleotides, etc.
Buffers referred to in this disclosure include "Tris," "Hepes", and "PBS."
"Tris" is tris(hydroxymethyl)aminomethane, and for the purposes of the
preferred embodiments of this invention is used at about pH 7. "Hepes" is
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, also used here as a
buffer at about pH 7. Phosphate-buffered saline, or "PBS," is 10 mM sodium
phosphate and 0.9 wt.% NaCl, used as an isotonic physiological buffer at
pH 7.4.
A cell is any one of the minute protoplasmic masses which make up organized
tissue, comprising a mass of protoplasm surrounded by a membrane including
nucleated and unnucleated cells and organelles. An intact cell is a cell
with an intact membrane that has not released its normal intracellular
components such as enzymes, organelles, or genetic material. A viable cell
is a living cell capable of carrying out its normal metabolic functions.
A complex (or a liposome complex) is defined as the product made by mixing
pre-formed liposomes comprising a compound of Formula I with a polyanion
(e.g., polynucleotide) or some other macromolecule containing multiple
negative charges. Such a complex is characterized by an interaction
between the polyanion and lipid components that results in the elution of
the polyanion and liposome together as substantially one entity through a
gel filtration column that separates on the basis of the Stokes' radius or
by some other separation procedure.
A charge ratio refers to a quantitative relationship between the net
positive charges contributed by the lipid and the net negative charges
contributed by the polyanion in a complex. The charge ratio herein is
expressed as positive to negative, i.e., 5:1 means five net positive
charges on the lipid per net negative charge on the polyanion.
Double-coated complexes are prepared from liposome complexes bearing a net
positive charge. Liposome complexes bearing a net positive charge are
prepared as described in the preceding paragraph, using a greater molar
amount of positively charged lipid than the molar amount of negative
charge contributed by the polyanion. These positively charged complexes
are mixed with negatively charged lipids to produce the double-coated
complexes. If sufficient negatively-charged lipid is added, the final
complex has a net negative charge. This definition includes liposomes that
have further modifications on the surface, such as the incorporation of
antibodies or antigens therein.
DOTMA is the most preferred lipid of Formula I, known as
N-(2,3-di(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammonium
chloride. DOTMA vesicles are liposomes made from DOTMA.
DNA represents deoxyribonucleic acid, which may optionally comprise
unnatural nucleotides. DNA may be single stranded or double stranded.
Drug refers to any therapeutic or prophylactic agent other than a food
which is used in the prevention, diagnosis, alleviation, treatment, or
cure of disease in man or animal. (Therapeutically useful polynucleotides
and polypeptides are within the scope of this definition for drugs).
Intracellularly means the area within the plasma membrane of a cell,
including the cytoplasm and/or nucleus.
A lipid of Formula I is to be understood as the class of lipids set forth
in the Summary of the Invention. Exemplary cyclic structures represented
by two or three of R.sup.3, R.sup.4 and R.sup.5 are quinuclidino,
piperidino, pyrrolidino and morpholino.
A liposome formulation is a composition of matter including a liposome,
which includes a material encapsulated in the liposome, for diagnostic,
biological or therapeutic use.
A liposome-polyanion complex is a composition of matter produced by
contacting a solution of polyanion with a preparation of cationic
liposomes produced from a compound of Formula I (with optional co-lipids
as appropriate).
Optional or optionally means that the subsequently described event or
circumstance may or may not occur, and that the description includes
instances where said event or circumstance occurs and instances in which
it does not.
An optional co-lipid is to be understood as a structure capable of
producing a stable liposome, alone, or in combination with other lipid
components, and is preferably neutral, although it can alternatively be
positively or negatively charged. Examples of optional co-lipids are
phospholipid-related materials, such as lecithin,
phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, sphinogomyelin, cephalin,
cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate,
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPG),
dioleoylphosphatidylglycerol (DOPC), dipalmitoylphosphatidylglycerol
(DPPG), dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE) and
dioleoylphosphatidylethanolamine
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-mal). Additional
non-phosphorous containing lipids are, e.g., stearylamine, dodecylamine,
hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl
stereate, isopropyl myristate, amphoteric acrylic polymers,
triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty
acid amides, dioctadecyldimethyl ammonium bromide and the like.
A pharmaceutical formulation is a composition of matter including a drug,
for therapeutic administration to a human or animal.
A pharmaceutically acceptable anion is an anion which itself is non-toxic
or otherwise pharmaceutically acceptable and which does not render the
compound pharmaceutically unacceptable. Examples of such anions are the
halide anions, chloride, bromide, and iodide. Inorganic anions such as
sulfate, phosphate, and nitrate may also be used. Organic anions may be
derived from simple organic acids such as acetic acid, propionic acid,
glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid,
succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid,
benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid, ehtane
sulfonic acid, p-toluenesulfonic acid, and the like.
A polyanion is a biologically active polymeric structure such as a
polypeptide or a polynucleotide, wherein more than one unit of the polymer
bears a negative charge and the net charge of the polymer is negative.
A polynucleotide is DNA or RNA containing more than one nucleotide.
Polynucleotides are intended to include ppp(adenyl 2'.fwdarw.5').sub.n
adenylate, n.gtoreq.2, represented by 2-5A. A polynucleotide comprising
riboinosinic acid and ribocytidylic acid is called poly IC.
Polynucleotides are those that can be made by chemical synthetic
methodology known to one of ordinary skill in the art, or by the use of
recombinant DNA technology, or by a combination of the two.
A polypeptide is a biologically active series of two or more amino acids
coupled with a peptide linkage.
RNA represents ribonucleic acid which may optionally comprise unnatural
nucleotides. RNA may be single stranded or double stranded.
A suitable aqueous medium for forming liposomes from the dried lipid film
is to be understood as, for example, water, an aqueous buffer solution, or
a tissue culture media. For example, a suitable buffer is phosphate
buffered saline, i.e., 10 mM potassium phosphate having a pH of 7.4 in
0.9% NaCl solution. The pH of the medium should be in the range of from
about 2 to about 12, but preferably about 5 to about 9, and most
preferably about 7.
A suitable solvent for preparing a dried lipid film from the desired lipid
components is to be understood as any solvent that can dissolve all of the
components and then be conveniently removed by evaporation or
lyophilization. Exemplary solvents are chloroform, dichloromethane,
diethylether, cyclohexane, cyclopentane, benzene, toluene, methanol, or
other aliphatic alcohols such as propanol, isopropanol, butanol,
tert-butanol, iso-butanol, pentanol and hexanol. Mixtures of two or more
solvents may be used in the practice of the invention.
A stable transfectant is a living cell into which DNA has been introduced
and become integrated in the genomic DNA of that cell
Topical administration includes application to any surface of the body,
including ocular administration and administration to the surface of any
body cavities.
Transdermal administration is administration through the skin with a
systemic effect.
Transfection refers for the purposes of this disclosure to the introduction
of DNA or RNA into a living cell
Unnatural nucleotides include those which are commercially available or
which can be readily made by means known to those of ordinary skill in the
art.
"Z" refers to the cis form of the aliphatic radicals in Formula I.
The compounds of this invention may be prepared as a racemic mixture of
D,L-isomers or as the individual D or L isomer. Because of the
availability of D or L starting materials, certain of these compounds are
readily prepared as the individual isomer. However, unless the specific
isomer is designated, it should be understood that this invention covers
both the pure D- or L- isomers as well as the D,L-racemate.
Compounds of Formula I have one asymmetric site, (marked above as *), and
thus can exist as a pair of optical isomers. Individual isomers of
compounds of Formula I are named herein using the IUPAC R-S convention,
sometimes called the "sequence rule." A description of the R-S convention
may be found, for example, in "Introduction to Organic Chemistry" by A.
Streitwieser, Jr. and C. Heathcock, (Macmillan Pub. Co., New York, 1976),
pages 110-114. Where appropriate, the optical activity of a compound may
be indicated by (+) or a (-) for the individual isomers, or (.+-.) for the
racemic mixture, referring to the direction in which a solution of the
compound rotates a plane of polarized light. For the purposes of the
appended claims, it should be understood that racemic mixtures of the
compounds of Formula (I) as well as either isomer taken alone are within
the scope of this invention.
B. Utility
The compounds of Formula I are particularly useful in the preparation of
liposomes, but may be used in any of the many uses for which cationic
lipids find application. For example, they may be used in industrial
applications, in food or feeds, in pharmaceutical formulations,
cosmetological compositions, or other areas where lipids may be employed.
These compounds may also be used in cosmetology, for example, in makeups,
lipstick, eyeshadow material, fingernail polishes, body lotion,
moisturizing creams, and the like. They may also be used for application
to the hair, either alone or in combination with other materials, such as
in shampoos, hair conditioners, permanent wave formulations or hair
straighteners, or as components in hair creams, gels, and the like.
Of particular interest is the use of these compounds in pharmaceutical
formulations, particularly topical formulations such as ointments, gels,
pastes, creams, and the like; and more particularly for the preparation of
pharmaceutical formulations containing liposomes. The consistency of the
formulation depends on the amount of aqueous solution used to make the
formulation. In such formulations containing compounds of this invention,
drugs which are insoluble or only sparingly soluble themselves in aqueous
solutions can be solubilized so that a greater concentration of drug can
be presented to the body.
In pharmaceutical formulations, these compounds may be used in those
contexts where cationic lipids are acceptable for the formulation of
creams, pastes, gels, colloidal dispersions, and the like. For additional
information, reference is made to Remington's Pharmaceutical Sciences,
17th Edition, Mack Publishing Company, Easton, Pennsylvania (1985), or any
other standard treatise on pharmaceutical formulations.
Other aspects of this invention are directed to the finding that
formulations comprising the compounds of Formula I are useful for
achieving desirable intracellular delivery of specific biologically active
substances, such as nucleosides, nucleotides, oligo- and poly-nucleotides,
steroids, peptides and proteins, and other appropriate natural or
synthetic molecules or macromolecules. The intracellular delivery can be
into the cytoplasm, into the nucleus, or both. Such intracellular delivery
can be achieved in tissue culture and may be used as an aid in
transfecting cells with desired polynucleotide sequences (e.g.,
deoxyribonucleic acid, DNA) to aid in cloning of specific sequences. Thus,
formulations comprising: (1) compounds of Formula I, and (2) DNA or
complementary DNA (cDNA) --in appropriate plasmids containing promoters,
enhancers and the like as desired--, can be utilized to achieve
transfection of cells and to obtain stable transfectants as part of the
process of cloning (via recombinant DNA technology well known to those
familiar in the art) various desired sequences to yield the corresponding
expressed products (e.g., proteins and peptides). The technology of
utilizing a compound of Formula I or other positively-charged lipid
formulation to achieve efficient transfection and to obtain stable
transfectants with the desired DNA sequences can enhance the ability to
achieve the desired end result of the cloning procedure. This technology
provides a less toxic and more efficient route for the delivery of
polynucleotides to cells than other presently-used techniques such as
calcium phosphate precipitation.
Intracellular delivery can also be achieved in the whole organism and may
be useful in several diverse applications. For example, enzyme-replacement
therapy can be effected by direct intracellular introduction of the
desired enzymes, or by appropriate transfection of cells with a DNA
sequence encoding the desired protein, with the appropriate promoters and
the like included so as to give sufficient gene expression. If desired,
inducible promoters can be employed to allow control in turning on or
turning off the gene of interest. Other applications of intracellular
delivery that can be achieved employing the compounds of Formula I or
other positively-charged lipid formulations for transfection of DNA
include but are not limited to hormone replacement therapy (e.g., insulin,
growth hormone, etc.), blood coagulation factor replacement therapy,
replacement therapy for other blood disorders such as .beta.-thalassemia
or other hemoglobin deficiencies, adenosine deaminase deficiency,
neurotransmitter replacement therapy, and the like. Another application
utilizing such formulations to enhance intracellular delivery includes the
delivery of "antisense" RNA oligomers to selectively turn off expression
of certain proteins. Compounds of this invention can also be used to
deliver biologically active materials across the blood brain barrier.
Formulations comprising the compounds of Formula I can also be used to
transfect and transform cells in vitro to introduce a desired trait before
implantation of the transformed cells into the whole organism. An example
of this application is to transfect bone marrow cells with a desired gene,
such as one coding for normal adult hemoglobin sequences to correct the
deficiency in patients with disorders such as .beta.-thalassemia,
adenosine deaminase deficiency, and sickle-cell anemia. The bone marrow
cells can be transfected in vitro, and then the appropriately transfected
cells can be transfused into the marrow of the patient. Alternatively, the
cells can be transfected in vivo as described herein. Procedures such as
calcium phosphate precipitation are much less efficient in effecting such
transfections, making them unsuitable for practical use. Other means of
achieving transfection that have been applied in vitro include the use of
viral vectors (such as SV-40 and retroviruses). However, these viruses are
oncogneic and thus cannot be safely used for transfecting cells in vivo or
in vitro for ultimate transfusion in vivo.
Intracellular delivery utilizing formulations of compounds of Formula I is
also useful for delivery of antiviral compounds (such as protease
inhibitors, nucleoside derivatives, nucleotides, or polynucleotides such
as 2-5A); anticancer compounds (including but not limited to
nucleosides/nucleotides such as 5-fluorouracil, adenosine analogs,
cytosine analogs, and purine analogs); antibiotics such as anthracyclines
(for example adriamycin and daunomycin) and bleomycin; protein antibiotics
such as neocarzinostatin, marcomomycin, and auromomycin; alkylating agents
such as chlorambucil, cyclophosphamide, nitrosoureas, melphalan,
aziridines, alkyl alkanesulfonates; platinum coordination compounds;
folate analogs such as methotrexate; radiation sensitizers; alkaloids such
as vincristine and vinblastine; cytoskeleton-disrupting agents;
differentiating agents; and other anticancer agents. This aspect of the
invention can be particularly useful in overcoming drug resistance such as
caused by reduced uptake mechanisms of the drug by the cells.
Further selectivity can be achieved by incorporating specific molecules
such as antibodies, lectins, peptides or protein, carbohydrates,
glycoproteins, and the like, on the surface of the liposome vesicles,
which can then serve to "target" the drugs formulated with the compounds
of Formula I to desired tissues bearing appropriate receptors or binding
sites for the ligand attached to the vesicle surface. Further selectivity
can also be achieved by coating the liposome vesicles with a neutral or
negatively-charged optional co-lipid (to eliminate non-specific adsorption
to cells) before addition of the targeting ligand as described above.
The use of formulations comprising compounds of Formula I or other
positively-charged lipid formulations of polynucleotides (including DNA
and RNA) for intracellular delivery is superior than other available
methodology, such a calcium phosphate coprecipitation, or polylysine or
DEAE-dextran complexation of polynucleotides, as the formulations of this
invention are much less toxic and deleterious to the living cells than are
the other above mentioned procedures. Furthermore, the formulations using
compounds of Formula I are much more efficient in transfecting cells.
Additionally, the use of liposomers made from the compounds of Formula I
to effect intracellular delivery of the liposome contents is superior to
the use of polyethyleneglycol (PEG) or glycerol-induced fusion of ordinary
neutral or negatively-charged vesicles to cells, because the vesicles of
the compounds of Formula I do not require the use of the PEG or glycerol
as fusion-inducing agents. These agents are highly deleterious to the
viablility and integrity of cells.
Another method that has been employed to induce fusion of liposomes with
cells involves incorporation of viral fusion proteins (such as the fusion
protein from Sendai virus) on the liposome surface. However, such
techniques are not only tedious but they also can result in formation of
antibodies by the animal against the viral proteins, thus severely
limiting the utility of this approach.
Other applications of the formulations of this invention comprising the
compounds of Formula I relate to localized delivery of drugs through the
stratum corneum, and to transdermal delivery of drugs. Liposome vesicles
comprising the compounds of Formula I can serve to introduce certain
compounds into and through the stratum corneum. Depending on the degree of
penetration enhancement (which is also influenced by the drug and the
incorporation of other components in the liposome, such as phospholipid
bilayer perturbing agents such as phosphatidylethanolmaine, Azone.RTM.,
and lysolecithin), the formulations can serve to enhance a localized
effect of the drug. This enhancement would be applicable to the treatment
of a localized outbreak of herpes simplex virus type 1 or 2 with an
interferon or an interferon inducer, and/or with a nucleoside such as an
acyclic guanosine nucleoside analog such as acyclovir or
9-(1,3-dihydroxy-2-propoxymethyl)guanine, or
9-(1,3-dihydroxy-2-propoxymethyl)guanine dipalmitate. In other cases, the
liposomes comprising compounds of Formula I can serve to enhance systemic
uptake of the drug by transdermal absorption, for example as with topical
applications of Synalar.RTM. in DOTMA formulations.
Another application of certain formulations comprising the compounds of
Formula I is the enhancement of a specific immune response, such as
humoral and/or cellular immunity, to an antigen of interest which is
incorporated in the lipid-containing vesicles. Thus, such preparations can
serve as specific adjuvants for vaccines (including viral, bacterial,
rickettsial, parasitic, and cancer vaccines), antigen preparations, as
well as other proteins or peptides including synthetic peptides of
interest. Additional components may be included to further enhance the
immune response, e.g., immunostimulants such as muramyl dipeptide/analogs.
N-acetylmuramyl-L-threonyl-D-isoglutamine may be particularly useful here.
C. Dosage and Administration
Administration of the active compounds and salts described herein can be
via any of the accepted modes of administration for the biologically
active substances that are desired to be administered. These methods
include oral, topical, parenteral, ocular, transdermal, nasal, and other
systemic or aerosol forms.
Depending on the intended mode of administration, the compositions used may
be in the form of solid, semi-solid or liquid dosage forms, such as, for
example, tablets, suppositories, pills, capsules, powders, liquids,
suspensions, or the like, preferably in unit dosage forms suitable for
single administration of precise dosages. The compositions will include a
conventional pharmaceutical carrier or excipient and an active compound of
Formula I or the pharmaceutically acceptable salts thereof and, in
addition, may include other medicinal agents, pharmaceutical agents,
carriers, adjuvants, etc.
Topical formulations composed of compounds of Formula I, other lipid
material, other penetration enhancers, phosphatidylethanolamine and
biologically active drugs or medicaments can be applied in many ways. The
solution can be applied dropwise, from a suitable delivery device, to the
appropriate area of skin or diseased skin or mucous membranes and rubbed
in by hand or simply allowed to air dry. A suitable gelling agent can be
added to the solution and the preparation can be applied to the
appropriate area and rubbed in Alternatively, the solution formulation can
be placed into a spray device and be delivered as a spray. This type of
drug delivery device is particularly well suited for application to large
areas of skin, to highly sensitive skin or to the nasal or oral cavities.
For oral administration, a pharmaceutically acceptable non-toxic
composition is formed by the incorporation of any of the normally employed
excipients, such as, for example pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose,
glucose, sucrose, magnesium, carbonate, and the like. Such compositions
take the form of solutions, suspensions, tablets, pills, capsules,
powders, sustained release formulations and the like. The exact
composition of these formulations may vary widely depending on the
particular properties of the drug in question. However, they will
generally comprise from 0.01% to 95%, and preferably from 0.05% to 10%,
active ingredient for highly potent drugs, and from 40-85% for moderately
active drugs.
Parenteral administration is generally characterized by injection, either
subcutaneously, intramuscularly or intravenously. Injectables can be
prepared in conventional forms, either as liquid solutions or suspension,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. Suitable excipients are, for example, water,
saline, dextrose, glycerol, ethanol or the like. In addition, if desired,
the pharmaceutical compositions to be administered may also contain minor
amounts of non-toxic auxiliary substances such as wetting or emulsifying
agents, pH buffering agents and the like, such as for example, sodium
acetate, sorbitan monolaurate, triethanolamine oleate, etc.
The amount of active compound administered will of course, be dependent on
the subject being treated, the type and severity of the affliction, the
manner of administration and the judgment of the prescribing physician. In
addition, if the dosage form is intended to give a sustained release
effect, the total dose given will be integrated over the total time period
of the sustained release device in order to compute the appropriate dose
required. Although effective dosage ranges for specific biologically
active substances of interest are dependent upon a variety of factors, and
are generally known to one of ordinary skill in the art, some dosage
guidelines can be generally defined. For most forms of administration, the
lipid component will be suspended in an aqueous solution and generally not
exceed 30% (w/v) of the total formulation. The drug component of the
formulation will most likely be less than 20% (w/v) of the formulation and
generally greater than 0.01% (w/v).
In general, topical formulations using a compound of Formula I are prepared
in gels, creams or solutions having an active ingredient in the range of
from 0.001% to 10% (w/w), preferably 0.01% to 5%, and most preferably
about 1% to about 5%. (Of course, these ranges ar | | |
