Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Cationic Amphiphile Micellar Complexes
The present invention relates to novel micellar complexes of cationic
amphiphilic compounds that facilitate delivery (andlor transfection) of
biologically active molecules to the targeted cells of a mammal. More
particularly, the present invention relates to the unique properties of these
micellar complexes and the methods of making and using micelles of cationic
amphiphiles to enhance delivery of biologicaily active molecules to the
desired cells of a mammal. A goal of the invention is to provide novel
complexes that can be used in gene therapy. The invention also relates to
the use of targeting agents that facilitate delivery of a biologically active
molecule to a specific; type of mammalian cell.
The effective introduction of foreign genes and other biologically active
molecules into targeted mammalian cells is a challenge still facing those
skilled in the art. Gene therapy requires successful transfection of target
cells
in a patient. Transfe~:.tion, which is practically useful per se, may
generally be
defined as a process of introducing an expressible polynucleotide (for
example a gene, a ct~NA, or an mRNA) into a cell. Successful expression of
the encoding polynuc;leotide thus transfected leads to production in the cells
of a normal protein and is also practically useful per se. A goal, of course,
is
to obtain expression sufficient to lead to correction of the disease state
associated with the abnormal gene.
Examples of diseases that are targets of gene therapy include:
inherited disorders such as cystic fibrosis, Gaucher's disease, Fabry's
disease, and muscular dystrophy. Representative of acquired target
disorders are: (1) for cancers--multiple myeloma, feukemias, melanomas,
ovarian carcinoma and small cell lung cancer; (2) for cardiovascular
conditions-progresai~e heart failure, restenosis, and hemophilias; and (3) for
neurological conditions-traumatic brain injury.
Cystic fibrosi~~, 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").
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Cystic fibrosis is characterized by chronic sputum production, recurrent
infections and lung destruction (Boat, T.F., McGraw Hill, inc., 1989, p. 2649-
2680). Though it is not precisely known how the mutation of the CFTR gene
leads to the clinical manifestation (Welsh, M.J. et al. Cel173:1251-1254,
1993), defective CI- sE;cretion and increased Na+ absorption (Welsh, M.J. et
al., Cel173:1251-1254., 1993; Quinton, P.M., FASEB Lett. 4:2709-2717,1990)
are well documented. Furthermore, these changes in ion transport produce
alterations in fluid transport across surface and gland epithelia (Jiang, C.
et
al., Science 262:424-~t27, 1993; Jiang, C. et al., J. Physiol. (London),
509.3:637-647, 1997; Smith, J.J. et al. J. Clin.l 9nvest., 91:1148-1153, 1993;
and Zhang, Y. et aB., Am.J.Physiol 270:C1326-1335, 1996). These resultant
alterations in water and salt content of airway surface liquid (ASL) may
diminish the activity of bactericidal peptides secreted from the epithelial
cells
(Smith, J.J. et al., Cell, 85:229-236, 1996) and/or impair mucociliary
clearance, thereby promoting recurrent lung infection and inflammation.
Several lines of evidence suggest that submucosal glands contribute to
the pathophysiology of CF lung disease. Maintenance of mucociliary
clearance requires the coordinate regulation of ciliary motion, ASL depth, and
mucin content. The quantity and composition of ASL are controlled by both
the epithelium and submucosal glands and based on estimates of cell volume
it appears that the latter may be a more important source of mucous
secretions. Recent studies also indicate that the serous cells of the
secretory
tubules of the submucosal glands are the predominant site of CFTR
expression in human bronchus and that fluid secreted from serous cells
flushes out mucins sE:creted by mucous cells. Additional evidence suggesting
that submucosal glands contribute to the pathophysiology of CF lung disease
includes: {1) CFTR is~ predominantly expressed in the serous cells of the
submucosal glands (Engelhardt, J.F. et al., Naf.Genef. 2:240-248, 1992), {2)
tracheal submucosal gland cultures from CF patients fail to secret CI-
(Finkbeiner, W.E., et al., Am.J.Physiol. 267:L-206-L-210,1996; Yamaya, M.,
et al., Am.J.Physioi. 261:L-485-L-490, 1991; Yamaya, M.; et al., .
Am.J.Physiol. 261:L-491-L-494,1991), (3) more than 60% of submucosal
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gland cultures from non-CF subjects showed a baseline secretion whilst
cultures from CF patients exclusively absorbed fluid (Jiang, C., et al., J.
PhysioL (London), 50 ~I.3:637-647, 1997), (4) obstruction of submucosal gland
ducts is the first pulmonary manifestation in CF patients, and is followed by
marked hyperplasia and hypertrophy (C?ppenheimer, E.H. et al., New York:
Year Book Medical Publishers, '1975, p. 241-278).
The evidence implicating submucosal glands in CF pathogenesis
suggests that effectivE: gene therapy for CF lung disease should target these
structures. Though numerous attempts have been made to transfer the CFTR
gene to surface epithelium, little attention has been paid to the submucosal
gland cells. Additionally, while it has been demonstrated that low levels of
~i-
galactosidase expression following intratracheal administration of adenovirus
vectors were detectak>le in submucosal glands (Pilewski, J.M., et al.,
Am.J.Physiol. 268:L6:57-6fi5, 1995), gland transfection levels were lower than
for surface epithelium, and declined markedly with distance from the airway
lumen.
Effective introduction of many types of biologically active molecules
has been difficult and not all the methods that have been developed are able
to effectuate efficieni~ delivery of adequate amounts of the desired molecules
into the targeted cells;. The complex structure, behavior, and environment
presented by an intact tissue that is targeted for intracellular delivery of
biologically active molecules often interferes substantially with such
delivery.
Numerous methods, including viral vectors, DNA encapsulated in liposomes,
lipid delivery vehicles,, and naked DNA have been employed to deliver DNA
into the cells of marn~mals. To date, delivery of DNA in vitro, ex viva, and
in
vivo has been demonstrated using many of the aforementioned methods.
Though viral tn~ansfection is relatively efficient, the host immune
response frequently !posses a major problem. Specifically, viral proteins
activate cytotoxic T lymphocytes (CTLs) which destroy the virus-infected cells
thereby terminating gene expression in the lungs of in vivo models examined.
The other problem is diminished gene transfer upon repeat administration of
viral vectors due to t!he development of antiviral neutralizing antibodies.
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These issues are presently being addressed by modifying both the vectors
and the host immune system. Additionally, non-viral and non-proteinaceous
vectors have been gaining attention as alternative approaches.
Because 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. Compoundis having both such domains may be termed
amphiphiles, and many lipids and synthetic lipids that have been disclosed for
use in facilitating suclh intracellular delivery (whether for in vitro or in
vivo
application) meet this. definition. One group of amphiphilic compounds that
have showed particullar promise for efficient delivery of biologically active
molecules are cationic amphiphiles. 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 bialogically active molecules
including, for example,-negatively charged polynucfeotides such as DNA.
Examples of cationic amphiphific compounds that are stated to be
useful in the intracellular delivery of biologically active molecules are
found,
for example, in the following references, the disclosures of which are
specifically incorporated by reference. Many of these references also contain
useful discussions of the properties of cationic amphiphile that are
understood in the art: as making them suitable for such applications, and the
nature of structures, as understood in the art, that are formed by compiexing
of such amphiphiies with therapeutic molecules intended for intracellular
delivery.
(1) Felgner, et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987)
disclose use of positively-charged synthetic cationic lipids including N-
[1{2,3-
dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"), to form
Iipid/DNA complexes suitable for transfections. See also Felgner et al., The
Journal of Biological Chemistrw, 209(4), 2550-2561 (1994).
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(2) Behr et al., Proc. Natl. Acad. Sci.. USA 86, 6982-6986 (1989) disclose
numerous amphiphiles including dioctadecylamidologlycylspermine
("DOGS").
(3) U.S. Patent 5,283,185 to Epand et al. describe additional classes and
species of amphiphiles including 3[3 [N-(N',N' - dimethylaminoethane)-
carbamoyl] cholesterol, termed °'DC-chol".
(4) Additional compounds that facilitate transport of biologically active
molecules into cells are disclosed in U.S. Patent No. 5,264,618 to Felgner et
al. See also Feigner et al., The Journal of Biological Chemistry 269(4), pp.
2550-2561 (1994) for disclosure therein of further compounds including
"DfUIRIE" 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium
bromide, which is discussed below.
(5) 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 Felgneir et al., Methods (Methods in Enzymoiogy), 5,
67-75 (1993).
(6) Brigham, K.L., B. Meyrick, B. Christman, M. Magnuson, G. King and
L.C. Berry. In vivo tr;ansfection of marine lungs with functioning prokaryotic
gene using a liposome vehicle Am.J.Med.Sci. 298:278-281, 1989.
(7) Gao, X.A. and L. Huang. A novel cationic liposome reagent for
efficient transfection of mammalian cells. ,Biochem Biophys Res Commun
179:280-285, 1991.
{8) Yoshimura, K., M.A. Rosenfeld, H. Nakarnura; E.M. Scherer, A.
Pavirani, J.P. Lecocq and R.G. Crystal. Expression of the human cystic
fibrosis transmembrane conductance regulator gene in the mouse lung after
in vivo intratracheal plasmid-mediated gene transfer. NucLAcids Res.
20:3233-3240, 1992.
(9) Zhu, N., D. Liggitt, Y. Liu and R. Debs. Systemic gene expression after
intravenous DNA delivery into adult mice. Science 261:209-211, 1993.
(10) Solodin, I., C.S. Brown, M.S. Bruno, C.Y. Chow, E. Jang, R.J. Debs
and T.D. Heath. A novel series of amphiphilic imidazolinium compounds for
in vitro and in vivo gene delivery. Biochem. 34:13537-13544, 1995.
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{11) Lee, E.R., J. Marshall, C.S. Siegal, C. Jiang, N.S. Yew, M.R. Nichols,
J.B. Nietupski, R.J. Ziegler, M. Lane, K.X. Wang, N.C. Wan, R.K. Scheule,
D.J. Harris, A.E. Smith and S.H. Cheng. Detailed analysis of structure and
formulations of cationic: lipids for efficient gene transfer to the lung.
Hum. Gene Ther. 7:1701-1717, 1996.
Additionally, several recently issued U.S. Patents, the disclosures of
which are specifically incorporated by reference herein, have described the
utility of cationic amphiphiles to deliver poiynucleotides to mammalian cells.
(U:S. Patent No. 5,676;954 to Brigham et al. and U.S. Patent No. 5,703,055
to Felgner et al.)
Although the compounds mentioned in the above-identified references
have been demonstrated to facilitate the entry of biologically active
molecules
into cells, it is believed that the uptake efficiencies provided thereby could
be
improved to support numerous therapeutic applications, particularly gene
therapy. Additionally, it is saught to improve the activity of the above-
identified compounds so that lesser quantities thereof are necessary, leading
to reduced concerns about the toxicity of such compounds or of the
metabolites thereof.
Another class of cationic amphiphiles with enhanced activity is
described, for example, in U.S. Patent No. 5,747,471 to Siege! et al. issued
May 5, 1998, U.S. Patent No. 5,650,096 to Harris et al. issued July 22, 1997,
and PCT publicatian 1N0 98102191 published January 22, 1998, the
disclosures ofiwhich are specifically incorporated by reference herein. These
patents also disclose formulations of cationic amphiphiles of relevance to the
practice of the present invention.
White there arc: many cationic amphiphiles and viral vectors that have
produced enhanced activity, new methods of binding and targeting lipid and
non-lipid delivery vehicles to specific mammalian cells are still sought. A
highly desired factor iin using cationic amphiphiles and viral vectors for
gene
therapy, and other applications of in viv~, in vitro and ex vivo delivery of
biologically active molecules, is the ability to effectively target and bind
to
specific mammalian cells. To date, effective methods which target specific
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cell types have been lacking. The ability to target specific cells would
reduce
the dosage of cationic:, amphiphile, viral or other delivery vehicle complexes
needed to effectively treat a specific disease state thereby reducing the
toxicity problems which are a function of higher doses. Consequently,
methods of improving the efficiency and the quantity of biologically active
molecules delivered t:o a desired mammalian cell are desired to enhance the
viability of cationic arnphiphile complexes, viral vectors, and other delivery
vehicles as successful therapeutic treatments.
Accordingly, the present invention is directed to novel micellar
complexes that facifit.ate delivery of biologically active molecules to the
cells of
a mammal. The novel micellar complexes are comprised of a cationic
amphiphile, a biologically active molecule, a derivative of polyethylene
glycol
(PEG), and optionallsr, a neutral, positive, or negative co-lipid. These novel
micellar complexes can possess unique properties that are not observed for
traditional cationic arnphiphile complexes. For example, the novel micellar
complexes enable one skilled in the art to preferentially bind the micellar
complex to airway epithelial cells. It may also be possible for the skilled
artisan to preferentially bind the micellar complex to other specific cell
types
or to enable targeting of a specific mammalian cell for delivery by the
micellar
complex.
The present invention provides for the use of a cationic amphiphile to
form a mixed micelle; complex with a PEG derivative and optionally a co-lipid.
All cationic amphiphiles that are capable of facilitating intracellular
delivery of
biologically active molecules are useful in the practice of the invention.
Although the invention is not limited to the amphiphiles disclosed, numerous
examples of cationic. amphiphiles useful in the practice of the invention are
described in the previously referenced publications.
In the practice of the invention, a micellar complex may be provided
wherein the complex is effective for binding to airway epithelial cells. Not
to
be limited as to theory, it is believed that the micellar complexes
demonstrate
preferential binding .as compared to traditional lipid complexes because of
the
difference in the charge density of the miceliar complex.
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The micellar complexes of the present invention may also be provided
wherein the complex is substantially more homogeneous when compared to
the traditional lipid complexes. In other words, micellar complexes of the
present invention have a narrower size distribution curve than lipid complexes
prepared by traditional means.
The preferred micellar complexes of the present invention are also
more stable than traditional lipid complexes. A micellar formulation maybe
prepared the previous day and stored over night without any adverse affects.
In a further aspect, the invention provides for the improved efficiency of
binding between the cationic amphiphile and the biologically active molecule.
The improved efficiency of binding results in a higher amount or greater
"loading" of DNA per lipid present in a formulation. It is known in the art
that
PEG derivatives stabilize a traditional lipid:bioiogically active molecule
complex and prevent precipitation. However, the micellar complexes, which
contain a PEG derivative, are able to load more biologically active molecule
without precipitation i:han the traditional lipid bilayer complexes that also
contain a PEG derivative. In other words, more biologically active molecules
are associated with each cationic amphiphile in a micellar complex as
compared to cationic amphiphiles in traditional cationic amphiphile
complexes.
In a still further aspect, the invention includes a method of making a
micellar lipid complex comprising a cationic amphiphile, a biologically active
molecule, a PEG derivative, and optionally a co-lipid. The resulting complex
is homogeneous, stable and effective for binding to airway epithelial cells.
In
a preferred embodiment, the complex is effective for systemic delivery of a
biologically active molecule.
The invention also provides for a method of delivering a biologically
active molecule to a mammalian cell by administering a micellar complex.
Additionally, a method is provided to facilitate transfection of a gene to a
mammalian cell by administration of a micellar complex.
In a still further aspect of the invention, the micellar complexes may
also include a targeting agent that facilitates delivery of a biologically
active
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molecule to a specific. type of mammalian cell. The targeting agents are
effective for both lipid and non-lipid methods and the invention provides for
use of targeting agents in all lipid complexes, including both traditional and
micellar cationic amphiphiles, along with the use of targeting agents in viral
vectors including adenoviruses,and other methods that have been employed
in the art to effectuate: delivery of biologically active molecules into the
cells of
mammals.
The invention also provides for pharmaceutical compositions of
micellar complexes and pharmaceutical compasitions of other lipid and non-
lipid complexes with 'targeting agents. The micella:- complexes may be the
active ingredient in a pharmaceutical composition that includes carriers,
fillers,
extenders, dispersants, creams, gels, solutions and other excipients that are
common in the pharrnaceutical formulatory arts.
Additional features and advantages of the invention will be set forth in
the description which follows, and in part will be apparent from the
description, or may k~e learned by practice of the invention. The objectives
and other advantages of the invention will be realized and attained by the
method particularly pointed out in the written description and ciaims herein
as
well as the appended drawings.
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Brief Descri .~tion of the Drawings
Figure 1, depicts a procedure for the formulation of traditional lipid
compiexes (a) compared to micellar complexes with (b) and without (c) a co-
lipid.
Figure 2. depicts the size distribution of a traditional cationic lipid GL-
67:pDNA complex (a) (GL-67:pDNA (0.5:0.5} & GL-67:DOPE:DMPE-
PEG5000 (1:2:0.05)) compared to the size distribution of micellar complexes
((b} & (c)). (b) (GL-67:DMPE-PEG5000:pDNA (1.5:0.5:2)} represents the size
distribution of a micellar complex lacking the minimum amount of PEG
necessary to form thE: preferred homogeneous complex, while (c} (GL
67:DMPE-PEG5000:~pDNA (1.5:0.75:2)) depicts the size distribution of a
micellar complex prepared with a sufFicient amount of PEG.
Figure 3. depicts the size distribution of a traditional cationic lipid GL-
89:pDNA complex (a) (GL-89:pDNA (2:2) & GL-67:DOPE:DMPE-PEG5000
(1:1:0.005)) compared to the size distribution of micellar complexes ((b) &
(c)). (b) (GL-89:DM1'E-PEG5000:pDNA (1.5:0.0025:2)) represents the size
distribution of a micellar complex lacking the minimum amount of PEG
necessary to form the preferred homogeneous complex, while (c) (GL-
67:DMPE-PEG5000:pDNA (1.5:0.25:2)) depicts the size distribution of a
micellar complex prepared with a sufficient amount of PEG.
Figure 4. depicts the change in size distribution of a micellar cationic
lipid GL-67:pDNA complex as the amounts of co-lipid and PEG
(DOPE:DMPE-PEGSOOO} are increased. A minimum amount of PEG is
necessary to form the small homogeneous and stable micellar complexes.
In the present: invention, cationic amphiphile compounds of the prior art
are used in formulations containing a PEG derivative and optionally a co-
lipid.
The resulting formulations are complexed to one or more biologically active
molecules. The novel formulations exhibit unique and surprising properties
that are not found in traditional cationic amphiphile formulations, other
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cationic amphiphile formulations, and lipid carriers. An additional aspect of
the invention is the use of targeting agents in the new formulations. The
targeting agents facilitate delivery to specific mammalian cells. The practice
of the invention is not limited as to theory.
Traditional complexes of a cationic amphiphile, a PEG derivative, and
optionally a co-lipid acre well known in the art. These traditional complexes
are formed by preparing a lipid film of the cationic amphiphile, the PEG
derivative, and optionally the co-lipid. The lipid film is then hydrated in
aqueous media to form a lipid bilayer which is then complexed to a
biologically active molecule. Traditional cationic amphiphile complexes
formed via this method are normally 400-500 nm (nanometers) in diameter
and vary in size by 5~0% or greater.
A preferred embodiment of the present invention is a small,
homogenous, and si:able mixed micelle formulation or micellar complex. One
embodiment of the iiwention contemplates a micellar complex that exhibits
binding to airway epithelia cells, a property not found with traditional
cationic
amphiphile campfex~es. 1n the practice of the present invention a micellar
complex formulation that can have unique and surprising properties is prepared
via a new method. The micellar complex may preferably be prepared by
hydrating the cationiic lipid and adding the hydrated cationic lipid to the
PEG
derivative which has also been hydrated in order to form a micellar lipid
suspension. The micellar cationic Iipid:PEG:biologically active molecule
complex is prepared by adding the micellar cationic Iipid:PEG derivative
solution
to the biologically active molecule. The molar ratio of lipid:biologically
active
molecule and of cationic Iipid:PEG derivative may vary over a wide range and
will depend on the cationic lipid, PEG derivative, and biologically active
molecule that is being utilized. The ratios may also vary significantly as a
function of administration site and disease target. In an embodiment, the
molar
ratio of lipid:biologic:ally active molecule is 1:8. In a further preferred
embodiment the biologically active molecule is DNA.
A micellar complex may also be prepared with a neutral, positive, or
negative co-lipid as part of the formulation. The c~-lipid is formulated with
the
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PEG lipid as a lipid film and hydrated as a single solution or the co-lipid
can be
formulated alone as a lipid film and hydrated with PEG lipid. The cationic
lipid is
then added in hydrate form to the PEG lipid and co-lipid solution to form a
micellar lipid which may then be used to form a micellar complex with a
biologically active molecule. In an embodiment, the molar ratio of
iipid:biologically active molecule is 1:8. In a further preferred embodiment
the
biologically active molecule is DNA.
In the practice of the invention, a micellar lipid complex may be provided
wherein the complex is effective for binding to airway epithelial cells. Not
to be
limited as to theory, it is believed that the micellar I~pid complexes
demonstrate
preferential binding as compared to traditional lipid complexes because of the
difference in the charge density of the micellar complex.
The micellar complexes of the present invention may also be provided
wherein the complex is substantially more homogeneous when compared to the
traditional lipid complexes. In other words, in this embodiment, micellar
complexes of the prE;sent invention have a narrower size distribution curve
than
lipid complexes prepared by traditional means. For example, the size
distribution of a traditional lipid complex may vary by greater than 50%
depending on the lipid, the DNA, and the Iipid:DNA ratio. By comparison, the
size distribution of micellar complexes in accord with this embodiment may
only
vary by a maximum of about 20%.
In addition to being significantly more homogeneous, the preferred
miceliar complexes of the present invention may nit appreciably vary in size
upon the addition of more biologically active molecules to a complex. For
example, experiments were preformed in which the ratios in micellar complexes
of cationic lipid to pt)NA and of cationic lipid to PEG derivative to co-lipid
were
constant while the amount of pDNA that was a part of the micellar complexes
was increased (i.e., the pDNA was not free in solution). The size of the
preferred micellar complexes and their size distribution did not vary
significantly
as the amount of pDNA in the micellar complexes was increased.
The preferred micellar complexes of the present invention are also mare
stable than traditional lipid complexes. Many traditional lipid complexes
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experience storage and stability problems which require special storage
procedures or mixing of the formulation with the DNA to be delivered
immediately before administration to a mammal or to cells in vitro. For
example
traditional cationic lipids are known to degrade via transacylation reactions
unless stored under :cpecifc conditions. Many traditional lipid:DNA complexes
are also known to precipitate out of solution shortly after complex formation
therefore requiring a postponement of the preparation of the complexes until
immediately before use. A pharmaceutical product ~nrhich requires the
formulation to be made immediately before use is not very practical. Micellar
complexes of the present invention preferably do not precipitate out of
solution
shortly after formulat6on. For example, a micellar formulation may be prepared
the previous day and stored over night without any adverse affects. One of
ordinary skill in the a!rt may also vortex a micellar formulation without
observing
significant precipitation.
A minimum arnount of PEG lipid is preferred to form a stable,
homogeneous complex when the micellar lipid solution is added to the
biologically active molecule. The minimum amount of PEG needed is
dependent upon the specific combination of cationic lipid and PEG lipid
selected. Methods to determine the minimum amount of PEG required to form
the micellar complex may include but are not limited to: 1) Observation of the
lipid:bioiogically active molecule complex following addition of the micellar
lipid
to the biologically active molecule to verify that the suspension is clear to
opaque and lacks particulates; 2) Sizing of the micellar.lipid:biologically
active
molecule complex following preparation using a particle sizer in order to
determine whether tine particle population is substantially homogeneous with
regard to particle size; and 3} Analysis of the behavior of the biologically
active
molecule in the mice~llar complex in agarose gel electrophoresis. More detail
regarding the above mentioned methods can be found in the examples
enclosed herewith.
Another embodiment of the invention relates to micellar complexes that
are smaller in diarne;ter than traditional cationic amphiphile complexes and
remain small and stable throughout a wide range of Iipid:DNA and lipid:PEG
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ratios. A minimum amount of PEG derivative is preferred to form small,
homogeneous micelta~r complexes. In a preferred embodiment of the invention,
a micellar complex pr~spared with one or more cationic amphiphiles, one or
more PEG derivatives., a biologically active molecule, and optionally a co-
lipid, is
on average approximately 25 to 250 nanometers in diameter. However the size
of a miceilar complex is dependent on the cationic lipid or lipids employed,
the
PEG lipid or lipids, thE~ amount and size of the DNA, and the co-tipid, if
present.
Not to be IimitE:d as to theory, the charge density of the rnicellar
complexes is believed to be responsible for the preferred unique and
surprising
property of the complexes to bind to airway epithelial cells. It is believed
that
the higher charge density translates into a higher affinity for certain cell
membranes. Consequently, many of the miceliar complexes bind to airway
epithelial cells. A simple in vitro fluorescence experiment demonstrates that
micellar complexes appreciably bind to exposed airway epithelial cells.
Traditional complexes of cationic amphiphiles, normally 400-500 nm in
diameter, do not exhiibit appreciable binding in the same experiment.
The micellar complexes of the present invention are also believed to be
less toxic upon administration to a mammal than traditional cationic lipid
complexes. For example, when injected intravenously into mice, micellar
complexes prepared using cationic lipid GL-67 were less toxic than traditional
cationic lipid complexes also prepared using GL-67. The lower toxicity of the
micellar complexes does not significantly affect the complexes ability to
deliver
to tumor cells. In a preferred embodiment, the micellar complexes maintain a
comparable deposition in tumor cells and specifically tumor endothelial cells
while they are less toxic in regard to other cells of a mammal.
In another embodiment of the present invention, the micellar complexes
are coated by lipids i~r other compositions used in the pharmaceutical arts to
coat compositions and formulations. For example, mixing a micellar complex
with a further hydrophobic species, such as a neutral lipid mixture, may coat
the
outside of the complex without disturbing the complex. Not being limited as to
theory, it is desirably to use the cationic Iipid:PEG derivative to condense
DNA
efficiently. The resulting small, highly condensed package of DNA, e.g., the
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micellar complex, can then be surrounded by other species and lipids, such as
co-lipids or other PEt; derivatives, which could interact with the charged
surface
of the condensed DNA. This may protect andlor mask the cationic Iipid:DNA
from the immune system. Since much of the toxicity of Iipid:DNA complexes is
due to bacterial sequience recognition, coating may be a valuable tool to
reducing toxicity. Additionally, the coating may facilitate the inclusion of a
targeting agent allowing delivery of the complex to a specific tissue or cell
type.
In a preferred embodiment, the coated complex is delivered systemically.
further, coatings may be useful in order to eXtend the residence time of
a micellar complex in the blood stream or as a time-release mechanism. Other
coatings or uses of coatings known in pharmaceutical arts are within the
practice of the invention.
Cationic Amphiphiles for Use in Micellar Complexes
This invention provides for the use of any cationic amphiphile or cationic
lipid compounds, and compositions containing them, that are useful to
facilitate
delivery of biologically active motecules to cells. Amphiphiles that are
particularly useful facilitate the transport of biologically active
polynucleotides
into cells, and in particular to the cells of patients fob the purpose of gene
therapy.
A number of preferred cationic arr~phiphiles according to the practice of
the invention can be: found in IJ.S. Patents No. 5,747,471 & 5,650,09fi and
PCT publication WO 98J02191, the disclosures of which are specifically
incorporated by reference herein. in addition to cationic arnphiphile
compounds, these two patents disclose numerous preferred co-lipids,
biologically active molecules, irorrnuiations, procedures, routes of
administration,
and dosages.
In connection with the practice of the present invention, cationic
amphiphiles tend to have one or more positive charges in a solution that is at
or
near physiological pH. Representative cationic amphiphiles that are useful in
the practice of the invention are:
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. -120)
(GL - 89)
(GL - 67)
NHp
and other amphiphilEa as are known in the art including those described in
U.S.
Patent No. 5,747,471, the disclosure of which is specifically incorporated by
reference herein.
NHZ and,
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PEG Derivatives
As discussed above, it has been surprisingly determined that the stability
of cationic amphiphilE; compositions (both traditional and miceilar) can be
substantially improved by adding to such formulations small additional amounts
of one or more derivatized polyethylene glycol compounds. Such enhanced
performance is partic,ularfy apparent when measured by stability of cationic
amphiphile formulations to storage and manipulation.
PEG derivatives were originally used to stabilize traditional cationic
amphiphile formulations. Not to be limited as to theory, the use of PEG and
PEG derivatives enables one to use a higher ratio of lipid to DNA. Previous
attempts to prepare more concentrated lipid:pDNA complexes using traditional
formulations resulted in precipitation of the complexes, especially at
Iipid:pDNA
ratios for which the nnajority of the pDNA was bound to lipid. It was believed
that the precipitation observed at higher concentrations in traditional
formulations might b~e related to a phase separation of the cationic lipid
component from the non-bilayer lipid component. In an attempt to maintain the
traditional lipid formulations in a bilayer configuration, PEG-containing
lipids
were found to be effective in preventing precipitation of the complex at
higher
pDNA concentrations.
Only a small mole fraction of PEG-containing lipid was used to form
stable traditional formulations that did not precipitate at high
concentrations of
lipid and DNA. For example, at 1.6 moi % PEG-DMPE, cationic Iipid:pDNA
complexes could be stabilized at pDNA concentrations exceeding 20 mM. For
more information regarding use of PEG derivatives the following references are
specifically incorporated by reference. Simon J. Eastman et al., Human Gene
Therapy, 8, pp. 765-773 (1997); Simon J. Eastman et al. Human Gene Therapy,
p. 8, pp. 313-322 (1997).
It was subsequently determined that a PEG derivative could be also used
to prepare novel micellar formulations. The PEG containing formulations of the
micellar complexes can exhibit unique properties not found with traditional
formulations of cationic amphiphiles that also contain PEG derivatives
including
improvement in the affinity of the formulations to biologically active
molecules.
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The improved efficiency of binding results in a higher amount or greater
"loading" of DNA per (lipid present in a formulation. It is known in the art
that
PEG derivatives stabilize the fipid:biologically active molecule complex and
prevent precipitation. However, the micellar complexes, which contain a PEG
derivative, are able tc~ load more biologically active molecule without
precipitation than the traditional lipid bilayer complexes that also contain a
PEG
derivative. in other words, more biologically active molecules are associated
with each cationic amphiphile in a micellar complex as compared to cationic
amphiphiles in traditional cationic amphiphile complexes. For example, at a
0.125:1 molar ratio oi' amphiphile:pDNA all of the pDNA appears to be
associated with the micellar complexes. Traditional cationic amphiphile
complexes require a 0.75:1 to 1:1 molar ratio of amphiphile:pDNA to completely
bind all of the pDNA. The high affinity for pDNA of the micellar systems
enables
one to deliver much more pDNA using fewer cationic amphiphiles.
In regard to micellar complexes, a minimum amount of PEG lipid can
form a stable, homocleneous complex when the micellar lipid solution is added
to the biologically active molecule. According to the practice of the
invention,
any derivative of polyethylene glycol may be part of the formulation to
prepare a
micellar complex. Complexes have been prepared using a variety of PEG
derivatives and all of the PEG derivatives, at a certain minimum cationic
amphiphiie:PEG derivative ratio have been able to form smail, homogeneous
complexes. The micellar complexes remain stable and homogeneous through
a wide range of cationic Iipid:PEG and cationic Iipid:DNA ratios once the
minimum amount of PEG lipid to form the small, homogeneous complexes is
determined. The minimum amount of PEG to form the stable, homogeneous
complex may be routinely determined by the skilled artisan.
The minimum amount of PEG used is dependent upon the specific
combination of cationic lipid and PEG lipid selected. For example, cationic
lipids with an acyl chain (GL-89) are less likely to pr~:cipitate upon mixing
with
biologically active molecules than cholesterol-based lipids such as GL-67.
This
is not to suggest that cholesterol-based lipids such as GL-67 are ineffective,
but
only that a different ~~ratio of cationic Iipid:PEG derivative is used to form
stable,
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homogeneous, micelllar complexes. Consequently, one might choose a cationic
lipid that is stable enough to form a micellar formulation without the
presence of
a PEG derivative.
Derivatives of polyethylene glycol useful in the practice of the invention
include any PEG polymer derivative with a hydrophobic group attached to the
PEG polymer. Exarrnples would include PEG-DSPE, PEG-PE, PEG-DMPE,
PEG-DOPE, PEG-DI'PE, or PEG-ceramide. Not to be limited as to theory, it is
believed that preferrE:d PEG-containing lipids would be any PEG polymer
derivatives attached to a hydrophobic group that can stabilizelinteract with a
cationic lipid. Two hiighly preferred species thereof include
dimyristoylphosphatidylethanolamine (di C,a) {"DMPE"); and
distearoylphosphatid'ylethanolamine (di C,$) ("DSPE").
With respect to selection of the PEG polymer , it is a preferred
embodiment of the invention that the polymer be linear, having a molecular
weight ranging from 1,000 to 10,000. Preferred species thereof include those
having molecular weights from 1500 to 7000, with 2000 and 5000 being
examples of useful, .and commercially available sizes. In the practice of the
invention, it is convenient to use derivatized PEG species provided from
commercial sources, and it is noted that the molecular weight assigned to PEG
in such products oftE:n represents a molecular weight average, there being
shorter and longer molecules in the product. Such molecular weight ranges are
typically a consequence of the synthetic procedures used, and the use of any
such product is withlin the practice of the invention.
It is also withi'.n the practice of the invention to use derivatized-PEG
species that (1) include more than one attached phospholipid, or (2) include
branched PEG sequence, or (3) include both of modifications (1 ) and {2).
Accordingly, preferred species of derivatized PEG include:
(a) polyethylene glycol 5000-dimyristoylphosphatidylethanofamine, also
referred to as PEGc~;ooo~-~DMPE;
(b) polyethylene glycol 2000-dimyristoylphosphatidylethanolamine, also
referred to as PEG~;,ooo~~DMPE);
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{c) polyethylene gllycol 5000-distearoylphosphatidylethanolamine, also
referred to as PEG~5oooa DSPE); and
(d) polyethylene glycol 2000-distearoylphosphatidylethanolamine, also
referred to as PEG~ZOOO~-DSPE).
Certain phospholipid derivatives of PEG may be obtained from
commercial suppliers. For example, the following species: di C14:0, di C16:0,
di
C18:0, di C18:1, and 16:0!18:1 are available as average 2000 or average 5000
MW PEG derivatives from Avanti Polar Lipids, Alabaster, AL, USA, as catalog
nos. 880150, 880160, 880120, 880130, 880140, 880210, 880200, 880220,
880230, and 880240..
Selection of Co-lipids.
The use of co-lipids is optional. Depending on the formulation, including
neutral, positive, or negative co-lipids in the miceliar complex may
substantially
enhance delivery and transfection capabilities. Representative co-lipids
include
dioleoylphosphatidylfahanolamine ("DOPE°'), the species most commonly
used
in the art, diphytanoylphosphatidyiethanolamine, lyso-
phosphatidylethanolamines other phosphatidyl-ethanolamines,
phosphatidylcholines, lyso-phosphatidylcholines, phosphatidyl-inositol 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, although a ratio from 2:1 through
1:2
is usually preferable. Use of diphytanoylphosphatidylethanolamine is highly
preferred according i:o the practice of the present invention, as is use of
"DOPE"'.
According to the practice of the invention, preferred formulations may
also be defined in relation to the mole ratio of PEG derivative, however, the
preferred ratio will vary with the cationic amphiphile chosen. A
representative
preferred micellar formulation according to the practice of the present
invention
has the cationic amphiphile GL-67: co-lipid DOPE: PEG~ooo molar composition
ratio of about 1:1:0.25. In preferred examples thereof, the co-lipid is
diphytanoylphosphatidylethanolamine, or is DOPE, and the PEG derivative is a
DMPE or DSPE conjugate of PEG2ooo or PEGSOOO~
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Bioloc~'rcatlY Active Molecules
Biologically active molecules that can be useful in the practice of the
invention include, for example, genomic DNA, cDNA, mRNA, antisense RNA or
DNA, oligodeoxynucleotides, polypeptides and small molecular weight drugs or
hormones. In the practice of the invention, one skilled in the art can as a
matter
of routine experimentation determine which molecules will be effectively
delivered to a mammalian cell. It is well known in the art that once delivery
of a
biologically active me>lecule by a cationic amphiphile complex (or other lipid
or
non-lipid carriers) to .a mammalian cell is demonstrated, the choice of other
molecules for delivery is routine.
Targeting and Tarcetina Complexes
A further aspect of the invention is the use of targeting agents in any of
the methods that efff~ctuate the delivery of biologically active molecules
into the
cells of mammals. In a preferred embodiment, targeting agents are used with
both traditional and micellar cationic amphiphile formulations or viral
formulations such a:. viral vectors and adenoviruses. A targeting agent is
usually a molecule, peptide sequence, or large protein that preferentially
taegets
or binds to specific mammalian cells. Many targeting agents are molecules that
are well known in thE~ art. For example, Pertactin, a peptide containing the
RGD
sequence, preferentially targets and binds to airway epithelial cells. In the
practice of the invention, a targeting agent or ligand is attached to a
carrier
complex. It is well-known in the art that although a lone targeting agent will
target specific cells, attachment of a targeting agent to another entity will
often
alter or destroy the rrralecule's targeting activity. However; attachment of a
targeting agent to a PEG derivative does not always destroy targeting agent
activity. Therefore, attachment of a targeting agent to a micellar complex may
also preserve the agent's activity, and it is a preferred embodiment of the
invention to attach a targeting agent to a micellar complex.
Goupling of ai targeting agent to a cationic amphiphile complex,
adenovirus, or other carrier wilt enable specific targeting to desired
mammalian
cells. Advantageously, targeting to a desired mammalian cell will enable more
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efficient delivery of biologically active molecules and therefore increase
transfection of the targeted mammalian cell.
A lipid comple;~c coupled to a targeting agent may comprise any cationic
amphiphile as described above, a PEG derivative, a biologically active
molecule, and optionally a co-lipid. Additionally, there may be formulations
in
which the PEG derivative is not necessary and the targeting agent is coupled
directly to a cationic amphiphilelbiologically active molecule complex with
optionally a co-lipid. The lipid complex may be a micellar or traditional
lipid
formulation. The targeting agent may also be appended directly to the PEG
derivative, i.e., PEG-IDMPE. In a further embodiment, the targeting agent is
coupled to a cationic polymer or to a hydrophobic rr~oiety such as a lipid.
Any targeting .agent known in the art may be useful in the practice of the
invention. Preferred targeting agents include: Pertactin, a peptide containing
an
RGD sequence that targets airway epithelial cells; UDPIUTP, which targets the
P2U receptors including P2U receptors on cells in the airways; Lactose, which
targets endogenous lectins in airways and the liver; and Cyclic RGD peptide,
which targets tumor endothelial cells. Other preferred targeting agents
include
Penetratin, an amphiphilic peptide, lectins, agents tn target the LDL
receptor,
mannose-6-phosphate which targets the rnannose-6-PO4 receptor, and airway
specific single chain antibodies.
In a preferred aspect of the invention, peptide ligands can be
incorporated into the lipid:biofogically active molecule complexes to augment
the transfection activity of the gene transfer system or to improve binding to
airway epithelial cells.
Other examples of peptide targeting agents include HAV peptides and
CNP-22 peptides. HAV peptides are a series of peptides containing the
sequence of histidine, alanine and valine that modulate cadherin-mediated cell
adhesion. Not to be limited as to theory, cadherin complexes form cell-cell
adhesion to maintain tissue integrity and generate physical and permeability
barriers in the body. Cadherins have been- shown to regulate epithelial,
endothelial, neural and tumor cell adhesion. The cell adhesion is achieved
through interactions between the extraceliuar domains of cadherins between
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cells, and cytoplasmic domains of cadherin with the catenin proteins and the
actin cytoskeleton wiithin the cell. A tri-peptide of histidine, alanine and
valine
(HAV) is located in the extracellular and cytoplasmic domains of cadherin(s).
The HAV peptide is crucial for hemophilic interactions between cadherins, and
plays an important role in the interaction with actin cytoskeleton via the
catenin
proteins. HAV peptides may be linear or cyclic.
in the practicE; of the present invention, HAV peptides preferably bind to
and becomes internalized by epithelial cells in airways and therefore may be
utilized as targeting agents for delivering biologically active molecules to:
1 }
epithelial cells, far example, as a targeting agent to deliver the cystic
fibrosis
transmembrane conductance regulator (GFTR) gene to airway epithelial cells in
CF lung; 2) endothelial cells, far example, inhibiting angiogenesis around a
tumor by delivering a gene that can cause apoptosis for endothelial cells; 3)
neural cells; and 4) tumor cells. HAV may also be chemically conjugated with,
for example, 1) poly-~L-lysine or linearlbranched palyethylenimine or other
polypeptides or (X}-phosphatidylethanoiamine (X-PE; e.g., N-MPB-PE) through
linkers) and pDNA complexes with or without lipids; or 2) viral vectors
through
linkers, to deliver biologically active molecules more efficiently to targeted
cells.
One may also use conjugate, positively charged HAV for pre-treatment followed
by administration of negatively charged non-viral or viral vectors, or co-
administer the mixed complexes of conjugated, positively charged HAV with
negatively charged non-viral or viral vectors.
HAV peptide:c may also be used as cell adhesion regulators. Some HAV
peptides are more potent at disrupting cell-cell adhesion junctions, and some
are more potent at preventing the formation of cell-cell adhesion junctions.
Based on these functions, one may use the peptides alone, or combined with
tight junction disrupting agents, like EGTA or palmitoyl-L-carnitine or
dimethyl ~3-
cyclodextrin or methyl p-cyclodextrin or a-cyclodextrin to improve delivery of
a
biologically active molecule to: 1 ) epithelial sells, for example, delivery
of the
CFTR gene through cell-cell permeability barriers in airway epithelial of CF
lung
to enhance gene uptake on the cell basolateral membrane; 2) endothelial cells,
for example, delivery of genes through brain-blood barriers to tumor cells; 3)
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neural cells, for exannple, to increase vector migration; 4) tumor cells,
especially
solid tumors (e.g., melanomas) since many solid tumars develop internal
barriers that limit the gene delivery to inner cells or cells distant from the
injection site; and 5) muscle, liver or other whole organs by local injection
with
vector in order to increase vector migration.
C-type natriuretic peptide containing 22 amino acid (CNP-22) binds to
and activates the gu~anylate cyclase-B (GC-B) receptor, a transmembrane
receptor that contains intracellular guanylate cyclasQ domain. Nat to be
limited
as to theory, the regulator pathway of the peptide is thought to be mediated
predominantly through cyclic GMP (cGMP). In the lung, CNP-22 binding and
function may predominate in airway epithelial cells. Specific binding of CNP-
22
to airway epithelial cells in vivo has been demonstrated by the functional
ability
of CNP-22 to elevate: cGMP levels, active CFTR-dependent chiaride transport,
and stimulate ciliary beat frequency. Additionally, CNP-22 conjugated with 16
lysine (K16-CNP-22) binds to some types) of epithelial cells in mouse trachea,
and other airways.
In the practice: of the invention, CNP-22 may be used as a targeting
agent. For exmple, adenovirus vector (AdV) mediated gene transfer to mouse
trachea, other airways; and lung are increased in mice treated with K16-CNP-
22. Enhancement of cationic Iipid:pDNA mediated gene transfer to the lung is
also observed in mice treated with K16-CNP-22. Since CNP-22 andlor GC-B
receptor have also been identified in brain, uterusloviduct, small intestine,
colon
anct kidney, CNP-22 peptides may also be used to target these organs for
delivery of biologicallly active molecules.
It is also within the practice of the invention to chemically conjugate CNP
with 1 ) poly-L-lysine, linearlbranch polyethylenimine or other polypeptides
or
(X)-phosphatidylethanolamines (X-PE; e.g.: N-MPB-PE) through linker(s), and
make pDNA complexes with or without lipids, or 2) viral vectors through
linker(s), to deliver genes more efficiently to targeted cells. Conjugated,
positiveiy charged C~NP may also be used for pre-treatment followed by
administration of negatively charged non-viral or viral vectors, or co-
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administration of the mixed complexes of conjugated, positively charged CNP
with negatively charc,~ed non-viral or viral vectors.
In the practice; of the invention, traditional and micellar complexes
containing targeting agents may be formulated and administered in the same
manner and using the same methods as complexes without targeting agents.
Similarly, the ratio of cationic amphiphile:PEG derivative and cationic
amphiphile:bioiogica~lly active molecule would be dependent on the type of
lipid
used.
Preparation of pharrnaceutical compositions and methods of administration
The present invention provides far pharmaceutical compositions that
facilitate delivery arnd/or transfection of biologically active molecules.
Pharmaceutical compositions of the invention facilitate delivery of
biologically
active molecules into tissues and organs such as the gastric mucosa, heart,
lung, liver, and tumor vasculature, and solid tumors. Additionally,
compositions
of the invention facilitate entry of bioiagically active molecules into cells
that are
maintained in vitro, :;uch as in tissue culture.
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; (d) ribozymes; and
(e) low molecular meight biolagically active molecules such as hormones and
antibiotics.
Cationic amphiphile species, PEG derivatives, and ca-lipids of the
invention may be blended so that two or more species of cationic amphiphile or
PEG derivative or co-lipid are used, in combination, to facilitate entry of
biologically active molecules into target cells andlor into subcellular
compartments therE:of. Cationic amphiphiles of the invention can also be
blended for such use with amphiphiles that are known in the art. Additionally,
a
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targeting agent may be coupled to any combination of cationic amphiphile, PEG
derivative, and co-lipid.
Dosages of the pharmaceutical compositions of the invention will vary,
depending on factoirs such as half-life of the biologically-active molecule,
potency of the biola~gically-active molecule, half-life of the amphiphile(s),
any
potential adverse eiffects of the amphiphile(s) or of degradation products
thereof, the route of administration, the condition of the patient, and the
like.
Such factars 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 miceilar complexes and pharmaceutical compositions
containing micellar complexes of the invention. Such preparations can be
administered orally, intravenously, parenterally, topically, transmucosally,
or by
injection of a preparation into a body cavity of the patient, or by using a
sustained-release formulation containing a biodegradable material, or by
onsite
delivery using additional micelles, gels and liposomes. Nebufizing devices,
powder inhalers, and aerosolized solutions are representative of methods that
may be used to administer such preparations to the respiratory tract. It is
aiso
within the practice of the invention to use micellar complexes for systemic
delivery.
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 complex
of
the invention are capable of determination by those skilled in the
pharmaceutical art. 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 addition,ai aspect of the invention concerns the protonation state of
the cationic amphiphiles of the complexes of the invention prior to their
contacting the biologically active molecule for delivery, or prior to the time
when
said complex contacts a biological fluid. It is within the practice of the
invention
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to provide fully protonated, partially protonated, or free base forms of the
amphiphiles in order to form, or maintain, such therapeutic compositions.
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Examples
Example 1 - Preparation of Micellar and Traditional Cationic
Lipid:Biologiciatly ~~ctive Molecule Complexes
The following ~°xample outlines typical procedures used to prepare
a
cationic lipid micellar complex. Figure 1 is a schematic representation that
depicts a procedure for the formulation of traditional cationic lipid
complexes (a)
as compared to cationic lipid micellar complexes with (b) and without (c) a co-
lipid. The practice of the present invention is not limited to the procedures
disclosed herewith.
Preparation of cationic lipid: PEG lipid: pDNA micellar complex
(1 ) The micelilar cationic: iipid:PEG lipid solution was prepared as follows.
The cationic lipid was hydrated at four times the concentration of the desired
final cationic lipid concentration of the Iipid:pDNA complex (a typical but
not
exclusive range is 0.25-16 mM cationic lipid). The PEG containing lipid was
hydrated at four times the concentration of the desired final PEG lipid
concentration of the Iipid:pDNA complex {a typical but not exclusive range is
0.25-16 mM cationic lipid). {In regard to the PEG lipid, a full range of lipid
anchors has been utilized and the PEG head group may be any one of a variety
of sizes.) Once hydrated, the cationic lipid was added to the PEG lipid at a
1:1 {vol:vol) ratio. lMiile this is a typical method, it is not required as
long as the
desired ratio of cationic Iipid:PEG lipid is ultimately achieved. The plasmid
DNA
was diluted to two tirnes the desired final pDNA concentration of the
Iipid:pDNA
complex. The cationic Iipid:PEG lipid:pDNA complex was then prepared by
adding the micellar cationic:PEG lipid solution to the pDNA at a 1:1 ratio
{vol:vol).
(2) The micellar cationic lipid solution was also prepared with a co-lipid
as part of the formulation. This was done as indicated above in (1) except
that
the co-lipid was forrr~ulated with the PEG lipid as a lipid film and hydrated
as a
single solution or in an alternative procedure the co-lipid was formulated as
a
lipid film and hydrate>d with PEG lipid. The PEG:co- lipid solution can then
be
substituted for the PEG lipid above.
Analysis of the micellar complex
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A minimum amount of PEG lipid was preferably used to form a stable,
homogeneous complex when the rniceliar lipid solution was added to the
biologically active molecule. Additionally, the minimum amount of PEG needed
was dependent upon the specific combination of cationic lipid and PEG lipid
selected. Methods to determine the minimum amount of PEG used to form the
micellar complex may include but are nofi limited to:
1 ) The lipid:bialogically active molecule complex was observed following
addition of the mice:llar lipid to the biologically active molecule. When the
micellar complex w:as observed after preparation, the suspension was clear to
opaque and lacked particulates. If particulates were observed, the formulation
was lacking a minimum amount of PEG to farm the preferred stable,
homogeneous micc:llar complexes. By comparison, traditional cationic
lipid:pDNA complexes were generally opaque solutions that did not have
particulates in them.
2) The micellar lipid:biologically active complex was sized following
preparation using a~ particle sizer. When the micellar complex was sized
following preparation, the particle population was substantially homogeneous
with regard to particle size and more preferably was small (in a preferred
embodiment appro:Kimately 25- 250 nm in diameter). If the size population
contained large, heterogeneous particles, the minimum amount of PEG lipid
was not present in the formulation. By comparison, traditional cationic
Iipid:pDNA complexes generally yielded particles that were around 200-800 nm
in diameter. These. suspensions tend to be quite heterogeneous in size and the
size of the complex: depended heavily on the cationic lipid used in the
formulation. No traditional cationic lipid complexes were generally observed
which exhibited the small, homogeneous characteristics observed with the
micellar formulations.
3) The behavior of the biologically active molecule in the micellar
complex was analyzed in agarose gel electrophoresis. If a minimum amount of
PEG lipid to form a stable, homogeneous micellar complex was used, the
biologically active rnalecule migrated into the agarase gel in a manner
different
from that of the free: biologically active molecule (although it was possible
to
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visualize a population of "free"plasmid in addition to the complexed plasmid}.
If
the minimum amount of PEG lipid had not been used, the majority of the
plasrnid visualized in the gel either: 1) migrated like free pDNA or 2) was
retained in the well oi'the gel and therefore was not visible in the gel. More
than
one of these tests was done in order to lend confidence that the minimum
amount of PEG lipid lhad been used.
Analysis of the: micellar Iipid:pDNA complex by agarose gel
electrophoresis was performed by preparing a 0.7% agarose gel in Tris-borate
EDTA buffer pH 8Ø A volume of micellar lipid:pDNA complex which contained
from 0.25-1 erg of pDNA was loaded per well. The gel was run for
approximately 1 hour at 100 V. The geiwas then stained overnight in 1X SYBR
Green nucleic acid stain (Molecular Probes) or another stain in order to
visualize the pDNA in the gel.
Example 2 - Size Distribution of lVliceilar Complexes
In Figures 2 and 3 the size distribution of traditional cationic Iipid:pDNA
complexes (Fig. 2A & 3A} are compared to the size distribution of cationic
Iipid:pDNA complexes prepared via the micellar method (Fig 2B, 2C, 3B, & 3C}.
The size distribution of a complex was determined by quasi-elastic tight
scattering with a Malvern Zeta-Sizer 4. The complex was sized within 1 hour of
preparation and the complex was measured at the manufacturer's
recommended count: rate of 50-250 kilocounts per second (KCPS). If
necessary, the count rate of the sample was adjusted to the desired range of
50-250 KCPS by dilution with water.
For the results depicted in Figure 2A, a cationic Iipid:pDNA complex
utilizing cationic lipidl GL-C7 was prepared in the traditional method. See
U.S.
Patent Nos. 5,747,471 and 5,650,096, the disclosures of which are specifically
incorporated by reference herein. In brief, the cationic lipid formulation
containing the cationic lipid, co-lipid, and the PEG lipid was either 1) dried
down
to a lipid film from chloroform or 2} lyophilized from t-butanol:water (9:1,
vol:vol}.
The resulting preparation was then hydrated to two times the desired final
concentration of the three lipids in the complex using distilled water. The
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cationic Iipid:pDNA complex is prepared by adding an equal volume of lipid to
the pDNA followed by gentle mixing. The same procedure was followed
replacing GL-67 with GL-89 for the size distributions depicted in Figure 3A.
In Figures 2B and 2C, a cationic lipid:pDNA complex utilizing cationic
lipid GL-89 was prepared via the micellar method. First, the miceliar cationic
Iipid:PEG lipid solutions were prepared as follows. GL-89 was hydrated at four
times the concentration of the desired final cationic lipid concentration of
the
cationic Iipid:pDNA complex. The PEG containing lipid was also hydrated at
four times the concentration of the desired final PEG lipid concentration of
the
cationic Iipid:pDNA complex. ~nce hydrated, the cationic lipid was added to
the
PEG lipid at a 1:1 (vol:vol) ratio. The plasmid DNA was then diluted to two
times the desired final pDNA concentration of the cationic lipid:pDNA complex.
The cationic Iipid:PEG Iipid:pDNA complex was then prepared by adding the
micellar cationic Iipid:PEG lipid solution to the pDNA at a 1:1 ratio
(vol:vol). The
same procedure was followed replacing GL-67 with GL-89 for the size
distributions depicted( in Figure 3B and 3C.
The size distriibutions of the traditional cationic lipid complexes, as seen
in figures 2A and 3A, are quite large varying from 200 nm to 1000 nm, for
example, for GL-67. The size distribution of the traditional complexes does
not
vary significantly as a function of the cationic Iipid:pDNA ratio. In figures
2B &
3B a micellar complex is formed, however, a minimum amount of PEG lipid to
form the preferred stable, homogeneous micellar complexes is not present. As
a result, the size distribution in figures 2B & 3B extends to sizes of greater
than
400 nm. Finally, in figures 2C & 3C, the size distribution of the preferred
micellar complexes prepared with a sufficient amount of PEG lipid are
depicted.
The size distribution of the preferred micellar complexes is significantly
more
homogeneous than traditional cationic lipid complexes and also significantly
more homogeneous than micellar lipid complexes lacking an effective amount
of PEG lipid.
Another example of the difference in size distribution of micellar
complexes that are lacking an effective amount of PEG lipid and the preferred
micellar complexes of the present invention which contain an effective amount
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WO 99/65461 PCT/US99/13875
of PEG lipid is shown in figure 4. Once an effective amount of PEG lipid is
added the size distribution becomes significantly more homogeneous.
Examale 3 - Bindina~ of traditional and micellar cationic lipid complexes to
airway epithelial cells
The following f~xample examines the ability of both traditional and
micellar cationic Iipid:pDNA complexes to bind to the surface of polarized
normal human bronchial ainrvay cells.
Growth of polairized normal human epithelial cells at an air-liquid
interface
Cell culture flasks were coated with human collagen by dissolving human
collagen (Sigma, human placental collagen, #7521 ) to 50mg/100mL in 0.2%
glacial acetic acid. Once dissolved, the collagen solution is filtered through
a
0.45 Nm filter set-up. This concentrated, sterile stock may be stored for 6
months at 4 C. The :>olution was prepared for coating of the flasks by
diluting
collagen stock 1:5 (vnl:vol) in sterile distilled water several minutes prior
to use.
The appropriate volume of diluted collagen was placed into a flask (12 mL for
T75 flasks, 24 mL for T150 flasks, and 400;.~L far each Millicell-PCF insert)
and
left for at least 2 hrs (preferably overnight) at 4 C. Following incubation at
4 C,
collagen was remave;d from flaskslinserts and Left in a sterile hood to dry
for 6-
12 hours. The flasksc/inserts were rinsed twice with sterile phosphate
buffered
saline, pH 7.4 containing penicillin/streptomycin. These flasks may be kept at
room temperature for up to 6 months.
A vial of normal human bronchial epithelial ells (Clonetics) was thawed
rapidly and split into five T150 flasks which have been pre-coated with human
collagen as indicated above. Cells were grown in the flasks with DMEM
(GibcoIBRL):BEGM (Clonetics) 1:1 (vol:vol) media in a 5% C02 environment.
Cells were grown to 80-90% confluence. Cells were then placed in Millicell-PCF
inserts (200 pl @ 2 a; 105 cellsl200 NI media) which were pre-coated with
human
collagen as indicated above. Twenty four hours after seeding, media was
removed from the inaert interior arid media on the exterior of the insert was
replaced with fresh media. Cells were then maintained in the air-liquid
interface
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_3~~
condition by replacing the exterior media every other day. Approximately 5-7
days following the switch to the air-liquid interface condition, cells
developed a
high trans-epithelial resistance.
Examination of the binding of traditional and micellar complexes to the
surface of polarized normal human bronchial airway epithelial cells
Normal human bronchial airway epithelial cells were grown at an air-
liquid interface as described above. Cells were maintained at the air-liquid
interface for approxirnately five days or until a trans-epithelial resistance
of
approximately 1000f2lcm2 developed. The cells were then ready for use in the
binding experiment.
Plasmid DNA was labeled non-covalently with the fluorescent probe
Toto-1-iodide (Molecular Probes) at a 1:200 molar ratio of Toto-1:pDNA
according to the manufacturer's instructions. Micellar and traditional
Iipid:pDNA
complexes were prepared at 10 times the desired lipid:pDNA concentration to
be used in the experiment using Toto-1 labeled plasmid. The micellar complex
was prepared as described above. The traditional complexes were prepared by
hydrating the traditional lipid films with water to 20 times the final
experimental
lipid concentration desired. Labeled pDNA was prepared at 20 times the final
experimental DNA concentration desired. The lipid was added to the labeled
pDNA and allowed to complex for 15 minutes. The complexes were then
diluted 1:10 (vol:vol) in Optimem.
The complexEa (approx. 300u1) were added to the apical membranes of
the airway epithelial cells in the interior of the insert and allowed to bind
for 1
hour at 37 C in a 5°/~ COZ atmosphere. The complex was then aspirated
from
the cell surface; the cell surface was washed 3 times with 0.5 mL cold PBS;
fixed for 15 minutes in 2% paraformaldehyde in PBS; and washed once with 0.5
rnL cold PBS. The insert was then cut out from the: insert housing, placed on
a
slide, coversiiped, and mounted with Immunomount (Shandon-Lipshaw)
containing 2 pg/mL DAPi as a nuclear counterstain.
Robust binding of the micellar complexes to the apical surface of the
airway cells was generally observed at cationic fipid:pDNA ratios of 50:50,
75:50
pM. There was negligible binding of traditional complexes in the same
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environment at cationic Iipid:pDNA ratios of 50:50, 75:50 NM. This methodology
should be applicable to essentially any adherent cell line.
