Note: Descriptions are shown in the official language in which they were submitted.
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COCHLEATE DELIVERY VEHICLES
Portions of the subject matter disclosed
herein were supported in part by movies or grants
from the United States Government.
This is a continuation in part of WO 96/25942 filed 22 February, 1996, which
is a
continuation-in-part of U.S. Patent No. 5,840,707 filed 22 February 1995,
which is a
continuation-in-part of U.S. Patent No. 5,643,574 filed 4 October 1993.
FIELD OF THE INVENTION
The instant invention relates to cochleates
and use thereof to stabilize biologic molecules,
such as carbohydrates, vitamins, minerals,
' polynucleotides, polypeptides, lipids and the like.
Cochleates are insoluble stable lipid-divalent
cation structures into which is incorporated the
biologic molecule. Because cochleates can be
biologically compatible, cochleates can be
administered to hosts by conventional routes and
can serve to deliver the biologic molecule to a
targeted site in a host.
BACKGROUND OF THE INVENTION
Plain lipid cochleates (Figure 1) have been
described previously. Protein-cochleates or
peptide-cochleates have been described heretofore
and patented by the .instant inventors, as
intermediate structures which can be converted to
protein-lipid vesicles (proteoliposomes) (Figure 2)
by the addition of calcium chelating agents (see
U.S. Pat. No. 4,663,161 and U.S. Pat. No.
4 , 871, 488)~
Freeze-fracture
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electron micrographs of protein-cochleates
containing Sendai glycoproteins made by the DC
method show the rolled up lipid bilayer structures
with a ''bumpy" surface. Plain phospholipid
cochleates are smooth in that type of preparation.
The proteoliposomes resulting from
polypeptide-cochleates have been shown to be
effective immunogens when administered to animals
by intraperitoneal and intramuscular routes of
immunization (G. Goodasin-Snitkoff, et al. , J.
Immunol., Vol. 147, p.410 (1991); M.D. Miller, et
al., J. Exu. Med., Vol. 176, p. 1739 (1992)).
Further, when the glycoproteins of Sendai or
influenza ,virus are reconstituted by that method,
the proteoliposa~mes are effective delivery vehicles
for encapsulated proteins and DNA to animals and to
cells in culture (R.J. Mannino and S.
Could-Fogerite, Biotechn,~g~,es, Vol. 6, No. 1, pp.
682-690 (1988); S. Could-Fogerite et al., Gene,
Vol. 84, p. 429 (1989); M.D. Miller, et al., J.
Exp. Med., Vol. 176, p. 1739 (1992)).
It would be advantageous to provide a means
for stabilizing or preserving biologic molecules in
a form that is stable at room temperature, capable
of desiccation and is suitable for oral
administration. For example, it would be
beneficial to have a formulation for stabilizing
polynucleotides and which could be used for
delivering polynucleotidss to a cell. A formulation
comprised of drugs, nutrients and flavors would
also be beneficial for the stabilization and
delivery of the molecules to a cell.
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BU~MY OF TH8 INV8D1TION
Accordingly, it is an object of the instant
invention to provide a means for stabilizing
biologic molecules to yield a formulation with
prolonged shelf life, which can be made into powder
form and which later can be rehydrated to yield a
biologically active molecule.
It also is an object of the instant invention
to provide a formulation suitable for use as a
vehicle to administer a biologically active
molecule to a host. The formulation can be used to
deliver a biologic molecule to the gut for
absorption or to a targeted organ, tissue or cell.
A suitable biologic molecule is a
polynucleotide or a bioactive compound such as a
lipophilic drug.
other suitable biologic molecules are
polypeptides such as hormones and cytokines or
nutrients such as vitamins, minerals, and fatty
acids.
Yet other suitable biologic molecules are
essential oils which impart flavor.
Those and other objects have been obtained by
providing a cochleate formulation comprising the
following components:
a) a biologically relevant molecule
component to be stabilized or delivered,
b) a negatively charged lipid component,
and
c) a divalent cation component.
In a preferred embodiment, the cochleate
formulation is administered orally.
The instant invention further provides a
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cochleate formulation containing a polynucleotide
wherein said polynucleotide-cochleate comprises the
following components:
a) a polynucleotide component,
b) a negatively charged lipid component,
and
c) a divalent cation co~tponent.
The polynucleotide can be one which is
expressed to yield a biologically active
polypeptide or polynucleotide. Thus, the
polypeptide may serve as an immunogen or, for
example, have enzymatic activity. The
polynucleotide may have catalytic activity, for
example, be a ribozyme, or may serve as an
inhibitor of transcription or translation, that is,
be an antisense molecule. If expressed, the
polynucleotide would include the necessary
regulatory elements, such as a promoter, as known
in the art.
The instant invention further provides a
cochleate formulation containing a polypeptide,
wherein said polypeptide-cochleate comprises the
following components:
a) a polypeptide component
b) a negatively charged lipid component,
and
c) a divalent cation component.
A specific example is an insulin cochleate.
The instant invention also provides a
cochleate formulation containing a lipophilic drug,
wherein said drug-cochleate comprises the following
components:
a) at least one drug,
b) at least one negatively charged lipid
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component, and
c) at least one divalent cation
component.
Thus, the drug may be an inhibitor of viral
replication such as that used in the treatment of
HERPES (acyclovir), or one prescribed for it~s
antifungal effect on mycotic infections (miconazole
nitrate). The drugs may also be those with specific
targeted effects on different physiological systems
such as anesthetics (propofol) which effect the
nervous system, or immunosuppressants, such as
cyclosporin A, which inhibit immune cell function.
Other lipophilic drugs may also be selected from
the groups of anti-infectious, anti-cancer,
steroidal anti-inflammatory, non-steroidal
anti-inflammatory, tranquilizer, or vasodilatory
agents.
The instant invention further provides a
cochleate formulation containing a nutrient,
wherein said nutrient-cochleate comprises the
following components:
a) at least one nutrient,
b) at least one negatively charged lipid
component, and
c) at least one divalent cation
component.
Specific examples include vitamin A-,
polyunsaturated fatty acids- and mineral-
cochleates.
The instant invention further provides a
cochleate formulation containing a flavor, wherein
said flavor-cochleate comprises the following
components:
a) at least one essential oil or extract,
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b) at least one negatively charged lipid
component, and
c) at least one divalent cation
component.
Examples include flavor substances generally
associated with essential oils and extracts
obtained from botanical sources such as herbs,
citrus, spices and seeds. Oils/extracts are
sensitive to degradation by oxidation, and because
the processing of the natural oils and extracts
often involves multiettp operations, costs are
generally considered to be higher. The advantage of
an oil/extract-cochleate would be in the
stabilization of these otherwise volatile and
expensive flavor substances. Flavor-cochleates can
also be incorporated into consumable food
preparations as flavor enhancers.
The advantages of cochleates are numerous.
The cochleates have a nonaqueous structure while
not having an internal aqueous space, and therefore
cochleates:
(a) are more stable than liposomes because
the lipids in cochleates ate less susceptible to
oxidation;
(b) can be stored lyophilized which provides
the potential to be stored for long periods of time
at room temperatures, which would be advantageous
for worldwide shipping and storage prior to
administration;.
(c) maintain structure even after
lyophilization, whereas liposom~ structures are
destroyed by lyophilization;
(d) exhibit efficient incorporation of
biological molecules, particularly with hydrophobic
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moieties into the lipid bilayer of the cochleate
structure;
(e) have the potential for slow or timed
release of the biologic molecule in vivo as
cochleates slowly unwind or otherwise dissociate;
(f) have a lipid bilayer matrix which serves
as a carrier and is composed of simple lipids which
are found in animal and plant cell membranes, so
that the lipids are non-toxic, non-immunogenic and
non-inflammatory;
(g) contain high concentration of divalent
cation, such as, calcium, an essential mineral;
(h) are safe, the cochleates are non-living
subunit formulations, and as a result the
cochleates have none of the risks associated with
use of live vaccines, or with vectors containing
transforming sequences, such as life threatening
infections in immunocompromised individuals or
reversion to wild type infectivity which poses a
danger to even healthy people;
(i) are produced easily and safely; and
(j) can be produced as defined formulations
composed of predetermined amounts and ratios of
biologically relevant molecules, including
polypeptides, carbohydrates and polynucleotides,
such as DNA, lipophilic drugs, and nutrients such
as vitamins, minerals and fatty acids.
The advantages of oral administration also are
numerous. An oral route has been chosen by the WHO
Children's Vaccine Initiative because of ease of
administration. Oral vaccines are less expensive
and much safer to administer than parenterally
(intramuscular or subcutaneous) administered
vaccines. The use of needles adds to the cost, and
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also, unfortunately, in the field, needles are
often reused.
~EBCRIPTI~~ OF THE DRANINAB
Figure 1 is a schematic representation of a
plain lipid cochleate.
Figure 2 shows the structure of
polypeptide-lipid vesicles with integrated membrane
proteins.
Figure 3 summarizes the various alternative
procedures for the preparation of cochleates.
Figures 4(A) and 4(B) show serum antibody
titers in mine following oral administration of
influenza poiypeptide-cochleates.
Figure 5 is a graph showing the results of
oral administration of polypeptide-cochleates when
challenged with live virus.
Figure 6 is a graphic representation of serum
antibody titers in mice following oral
administration of Sendai-cochleates.
Figure 7 is a graph depicting the induction of
antigen-specific cytotoxic splenocytes following
oral administration of Sendai cochleates.
Figure 8 provides a series of bar graphs
depicting serum glucose levels before and after
oral insulin administration.
DL"fl~rIhED DESCRIPTIOI~T OF THE INVED1TION
The instant inventors have now found
surprisingly and have demonstrated that cochleates
themselves be used as means for stabilizing and
delivering biologic molecules. The cochleates
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survive the harsh acid environment of the stomach,
protecting the susceptible biologic molecules
immersed therein, probably by virtue of their
unique multilayered precipitate structure. It is
likely that cochleates then are taken up by
microfold cells (M cells) in the small intestine.
The instant inventors have demonstrated that
oral administration by drinking cochleates
containing the glycoproteins and viral lipids from
the surface of influenza or Sendai viruses plus
phosphatidylserine and cholesterol, stimulate both
mucosal and circulating antibody responses. In
addition, strong helper cell (proliferative) and
killer (cytotoxic) cell responses also are
generated. Perhaps most impressively, oral
administration of the influenza cochleates protects
against intranasal challenge with live virus.
Those results are unexpected for a number of
reasons.
It was not known and was nat expected that the
cochleates would survive the stomach and protect
the polypeptides associated with them from the acid
environment and degradative enzymes. It is known
that without the presence of at least 3 mM calcium,
the cochleates begin to unwind and form liposomes.
It was possible, in fact likely, that the
cochleates would not remain intact during the
transit from the mouth, down the esophagus and
through the stomach. If cochleates did come apart,
they would be digested as food.
Also, having survived the stomach, that the
cochleates would interact in an effective way with
the mucosal and circulating immune systems was
unknown and unexpected. Everyone ingests large
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quantities of proteins, fats and sugars on a daily
basis which simply get digested and used as fuel,
without stimulating any kind of mucosal or
circulating immune responses. Thus, the cochleates
deliver molecules Which retain biologic activity at
the delivery site within the host.
As used herein, the term "immune response"
means either antibody, cellular, proliferative or
cytotoxic activities, or secretion of cytokines.
Also, as used herein, the term "antigen" is
meant to indicate the polypeptide to which an
immune response is directed or an expressible
polynucleotide encoding that polypeptide.
"Polynucleotide" includes DNA or RNA, as well
as antisense and enzymatically active molecules.
Thus the biologically relevant molecule can be the
polynucleotide itself, the transcript thereof or
the translated polypeptide encoded thereby.
"Polypeptide" is any oligomer or polymer of
amino acids . The amino acids can be L-amino acids
or D-amino acids.
A "biologically relevant molecule" is one that
has a role in the life proces es of a living
organism. The molecule may be organic or
inorganic, a monomer or a polymer, endogenous to a
host organism or not, naturally occurring or
synthesized in vitro and the like. Thus, examples
include, vitamins, minerals, amino acids, toxins,
microbicides, microbistats, co-factors, enzymes,
polypeptides, polypeptide aggregates,
polynucleotides, lipids, carbohydrates,
nucleotides, starches, pigments, fatty acids, fatty
acids of polyunsaturated form, flavored essential
oils or extracts, hormones, cytokines, viruses,
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organelles, steroids and other multi-ring
structures, saccharides, metals, metabolic poisons,
drugs and the like.
The instant invention also can be practiced
using whole cells other subcellular replicative
entities, such as viruses and viroids. Hence,
bacteria, yeasts, cell lines, viruses and the like
can be mixed with the relevant lipid solution,
caused to precipitate to yield structures wherein
the cells and the like are fixed within the
cochleate structure.
Polypeptides are suitable molecules to be
incorporated with cochleates. The procedure for
preparing cochleates ie set forth in greater detail
hersinbelow. The polypeptide is suspended in a
suitable aqueous buffer. The lipids are dried to
form a thin film. Then the aqueous buffer is added
to the lipid film. The vessel is vortexed and then
the sample dialyzed against a cation-containing
buffer.
In that way, for example, cochleates carrying
insulin can be obtained. The insulin cochleates
were made with a 1 mg/ml solution of insulin, but
various other beginning concentrations of insulin
can be used to obtain cochleates loaded with
varying concentrations of insulin.
Recent studies indicate that the direct
injection of DNA plasmids can lead to the
expression of the proteins encoded by those
plasmids resulting in humoral and cell mediated
immune responses, see, for example, Wang et al.,
Proc. Natl Acad. Sci. 90: 4156'4160 (1993); Zhu et
al., Science 261: 209-211 (1993). Those studies
indicate that DNA vaccines could provide a safe and
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effective alternative for human vaccination. Those
studies also suggest that DNA vaccines could
benefit from simple, more efficient delivery
systems.
The use of lipids to facilitate the delivery,
entry and expression of DNA in animal cells is well
documented, see, for example, Philip et al., Col.
dell Biol. 14: 2411-2418 (1994). Indeed, DNA-lipid
complexes currently form the basis for a number of
human gene therapy protocols.
Because cochleates are stable structures which
can withstand a variety of physiologic conditions,
cochleates are suitable means for delivering
biologic molecules, such as, polypeptides or
polynucleotides, to a selected site in a host. The
polypeptide or polynucleotide is incorporated into
and integral with the cochleate structure. Thus
the polygeptide or polynucleotide, which may need
to be expressed, are protected from degrading
proteases and nucleases.
The cochl.eates used in the instant invention
can be prepared by known methods such as those
described in U.S. Patent No. 4,663,161, filed 22
April 1985, U.S. Patent No. 4,871,488, filed 13
April 1987, S. Could-Fogerite et al., Analytical
Biochemistry, Vol. 148, pages 15-25 (1985); S.
Could-Fogerite et al., ~ van es in Membrane
Biochemistry a_f~1 Bioen~~g~etics, edited by Kim,
C.H., Tedaschi, T., Diwan, J.J., and Salerno, J.C.,
Plenum Press, New York, pages 569-586 (1988); S.
Could-Fogerite et al., en , Vol. 84, pages
429-438 (1989); Litiosome Technoloav, 2nd Edition,
Vol. I, Liposome Preparation and Related
Techniques, Vol. II, Entrapment of Drugs and Other
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Materials, and Vol. III, Interactions of Liposomes
with the Biological Milieu, all edited by Gregory
Gregoriadis (CRC Press, Boca Raton, Ann Arbor,
London, Tokyo), Chapter 4, pp 69-80, Chapter 10, pp
167-184, and Chapter 17, pp. 261-276 (1993); and
R.J. Mannino and S. Gould-Fogerite, Liposome
Mediated Gene Transfer, Biotechniaues, Vol. 6, No.
1 (1988), pp. 682-690.
The polynucleotide can be one which expresses
a polypeptide, that is, pathogen membrane
polypeptides, aberrant or atypical cell
polypeptides, viral polypeptides and the like,
which are known or which are suitable targets for
host immune system recognition in the development
of immunity thereto.
The polynucleotide may express a polypeptide
which is biologically active, such as, an enzyme or
structural or housekeeping protein.
Also, the polynucleotide may be one which
necessarily is not expressed as a polypeptide but
nevertheless exerts a biologic effect. Examples
are antisense molecules and RNA's with catalytic
activity. Thu~c, the ~xpressed sequence may on
transcription produce an RNA which is complementary
to a message which, if inactivated, would negate an
undesired phenotype, or produce an RNA which
recognizes specific nucleic acid sequences and
cleaves same at or about that site and again, the
non-expression of which would negate an undesired
phenotype.
The polynucleotide need not be expressed but
may be used as is. Thus, the polynucleotide may be
an antisense molecule or a ribozyme. Also, the
polynucleotide may be an immunogen.
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Thus, for polynucleotides, the relevant coding
sequence is subcloned downstream from a suitable
promoter, other regulatory sequences can be
incorporated as needed, in a vector which is
expanded in an appropriate host, practicing methods
and using materials known and available in the art.
For examplt, two plasmids, pDOLHIVenv (AIDS
Research and Reference Reagent Program, Jan. 1991
catalog p. 113; Freed et al. J. Virol. 63: 4670
(1989)) and pCMVHIVLenv (Dr. Eric Freed, Laboratory
of Molecular Immunology, NJAID, NIH) are suitable
expression plasmids for use in
polynucleotide-cochleates.
The plasmids contain the open reading frames
for the env, tat and rev coding regions of HIV-1
(LAV strain).
pDOLHIVenv was constructed by introducing the
SalI-XhoI fragment from the full length infectious
molecular clone pNL4-3 into the SalI site of the
retrovirus vector, pDOL (Korman et al. ~~roc. Natl.
Acad. Sci. 84: 2150 (1987)). Expression is from
the Moloney murine virus LTR.
pCMVHIVLenv was constructed by cloning the
same SalI-XhoI fragment into the XhoI site of the
cytomegalovirus (CMV)-based expression vector p763.
The polynucleotide can be configured to encode
multiple epitopes or epitopes conjugated to a known
immunogenic peptide to enhance immune system
recognition, particularly if an epitope is only a
few amino acids in size.
To form cochleate precipitates, a majority of
the lipid present should be negatively charged.
One type of lipid can be used or a mixture of
lipids can be used. Phosphatidylserine or
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phosphatidylglycerol generally have been used.
Phosphatidylinositol also forms a precipitate which
converts to ligosomes on contact with EDTA. A
substantial proportion of the lipid can, however,
be neutral or positively charged. The instant
inventors have included up to 40 mol% cholesterol
based on total lipid present and routinely make
polypeptide-lipid or polynucleotide-lipid
cochleates which contain 10 mol% cholesterol and
2 0 % v i r a 1 m a m b r a n a 1 i p i d s .
Phosphatidylethanolamine, plain or cross-linked to
polypeptides, also can be incorporated into
cochleates.
While negatively charged lipid can be used, a
negatively charged phospholipid is preferred, and
of those phosphatidylserine, phoaphatidylinositol,
phosphatidic acid and phosphatidylglycerol are most
pref erred .
One skilled in the art can determine readily
how much lipid must be negatively charged by
preparing a mixture with known concentrations of
negative and non-negative lipids and by any of the
procedures described herein, determining whether
precipitates form.
Th~re are several known procedures for making
the cochleates of the instant invention and those
are schematized in Figure 3.
A suitable procedure for making cochleates is
one wherein a negatively charged lipid such as
phosphatidylserine, phosphatidylinositol,
phosphatidic acid or phosphatidylglycerol in the
absence or presence of cholesterol (up to 3:1,
preferably 9:1 w/w) are utilized to produce a
suspension of multilamellar lipid vesicles
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containing or surrounded by a biologically relevant
molecule (polypeptide, polysaccharide or
polynucleotide, such as DNA) which are converted to
small unilamellar protein lipid vesicles by
sonication under nitrogen. Alternatively, to avoid
daaage, the biologically relevant molecule can be
added to the solution following sonication. The
vesicles are dialyzed at room temperature against
buffered divalent cation, e.g., calcium chloride,
resulting in the formation of an insoluble
precipitate which may be presented in a form
referred to as a cochleate cylinder. After
centrifugation, the resulting pellet can be taken
up in buffer to yield the c~hleate solution
utilized in the instant invention.
In an alternative and preferred embodiment, an
amount of negatively charged lipid, e.g.,
phosphatidylserine and optionally, cholesterol in
the same proportions as above and equal to from
about 1 to 10 times the weight, preferably equal to
four times the weight of the viral or other
additional lipids (including polyunsaturated fatty
acids or essential oils) are utilized to prepare
the cochleates. Either a polypeptide, a mineral
such as calcium, magnesium, barium, iron or zinc, a
vitamin such as vitamins A, D, E or K, a lipophilic
drug, a flavor, a carbohydrate or polynucleotide,
such as DNA, is added to the solution. That
solution then is dialyzed against buffered divalent
cation, e.g., calcium chloride, to produce a
precipitate which can be called a DC (for direct
calcium dialysis) cochleate.
An additional, related method for
reconstituting cochleates has been developed and is
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called the LC method (liposomes before cochleates).
The initial steps involving addition of extracted
polypeptide, polysaccharide,polynucleotide, such as
DNA or combinations thereof, to dried down
negatively charged lipid and cholesterol are the
same as for the DC method. However, the solution
next is dialyzed against buffer (e.g., 2 mM TES, 2
mM L-histidine, 100 mM NaCl, pH 7.4) to form small
liposomes containing the polypeptide,
polynucleotide, such as DNA, and/or polysaccharide.
A divalent cation, e.g., calcium, then is added
either directly or by dialysis to form a
precipitate which can consist of cochleates.
In the above procedures for making the
cochleates of the instant invention, the divalent
cation can be any divalent cation that can induce
the formation of a cochleate or other insoluble
lipid-antigen structures. Examples of suitable
divalent cations include Ca;z, Mg+Z, Ba'2, and Zn~z or
Fe~2 other elements capable of forming divalent ions
or other structures having multiple positive
charges capable of chelating and bridging
negatively charged lipids.
Cochleates made with different cations have
different structures and convert to liposomes at
different rates. Because of those structural
differences, the rate of release of the
biologically relevant molecules contained therewith
varies. Accordingly, by combining cochleates made
with different cations, formulations which will
release the biologically relevant molecule over a
protracted period of time are obtainable.
The amount of biologically relevant molecule
incorporated into the cochleates can vary. Because
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of the advantageous properties of cochleates
generally, lesser amounts of biologically relevant
molecule can be used to achieve the same end result
as compared to using known delivery means.
An artisan can determine without undue
experimentation the optimal lipid: biologically
relevant molecule ratio for the targeted purposes.
Various ratios are configured and the progress of
precipitation of each sample is monitored visually
under a phase contrast microscope. Precipitation
to fona, for example, cochleates, is monitored
readily. Then, the precipitates can be
administered to the targeted host to ascertain the
nature and tenor of the biologic response to the
administered cochleates.
It should be evident that the optimized ratio
for any one use may range from a high ratio, for
example, to minimize the use of a rare biologically
relevant molecule, to a low ratio to obtain maximal
amount of biologically relevant molecule in the
cochleates.
Cochleates can be lyophilized and stored at
room temperature indefinitely or can be stored in a
divalent cation-containing buffer at 4°C for at
least six months.
The cochleate formulations also can be
prepared both with and without fusogenic molecules,
such as Sendai virus envelope polypeptides. Prior
studies with proteoliposomes have demonstrated that
cytoplasmic delivery of lipo:ome contents requires
a fusogenic liposome bilayer. The exact role of
Sendai virus enwelope polypeptides in facilitating
the immune response to polypeptide-cochleates as
yet is not clear.
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It is preferred to use cochleates without
fusogenic molecules over fu:ogenic molecule
cochleates because of a more simple structure and
ease of preparation favors eventual use in humans.
Because polynucleotides are hydrophilic
molecules and cochleates are hydrophobic molecules
that do not contain an internal aqueous space, it
is surprising polynucleotid~s can be integrated
into cochleates. The polynucleotida: are not
exposed on the surface of the cochleates because
the polynucleotides are resistant to nucleases.
In the case of polynucleotide cochleates,
considerations for dosage parallel the standard
methodologies regarding vaccines as known in the
art. Also, methods for using polynucieotides in
liposomes and the "nak~sd DNA" are available to
serve as a baseline for empirically determining a
suitable dosing regimen, practicing known methods.
For example, a suitable scheme for determining
dosing is as follows.
The initial dose of polynucleotides in
cochleates admin~.stered by injection to animals is
selected to be about 50 fig, although it is know
that as little as 2~g of tested plasmids is
effective. That dose is proposed to maximize the
probability of observing a positive response
following a single administration of a cochleate.
Any formulations which do not elicit a response at
that dose are to be considered ineffective but
retained for further study.
Developing formulations which can be
administered easily and non-invasively is
desirable. Thus, PO administration of cochleates
will be targeted and higher doses will be tried
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initially (100 ~Cg/animal and 200 ~,g/animal).
However, lower. doses are required for parenteral
routes.
Then graded doses will be used to develop a
dose response curve for each formulation. Thus,
cochleates containing 50 fig, l0 ~cg, 2 ~cg, 0.4 and 0
~g polynucleotide/animal will be inoculated with at
least l0 animals per group.
Immune response or enzymatic activity are
responses easily monitored when expression of the
polynucleotide is required. Altered phenotype is
another response for tracking efficacy of antisense
or ribozyme type molecules. In the case of immune
system monitoring, T cell proliferation, CTL and
antibody presence at specific body sites can be
evaluated, using known methods, to assess the state
of specific immune response.
To determine the duration of activity of
cochleate formulations, groups which have responded
to a single immunization are monitored periodically
for up to a year or more to determine the effective
life of a cochlea~te on administration.
Animals which fail to develop a detectable
response on first exposure can be re-inoculated
(boosted) to provide insights into the ability of
the low dose formulations to prime the immune
system for later stimulation.
Pharmaceutical formulations can be of solid
form including tablets, capsules, pills, bulk or
unit dose powders and granules or of liquid form
including solutions, fluid emulsions, fluid
suspensions, semisolids and the like. In addition
to the active ingredient, the formulation would
comprise suitable art-recognized diluents,
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carriers, fillers, binders, emulsifiers,
surfactants, water-soluble vehicles, buffers,
solubilizers and preservatives.
An advantage of the cochleates is the
stability of the composition. Thus, cochleates can
be administered orally or by instillation without
concern, as well as by the more traditional routes,
such as topical, subcutaneous, intradernal,
intramuscular and the like, Dirtct application to
mucosal surfaces is an attractive delivery means
made possible with cochleates.
The skilled artisan can determine the most
efficacious and therapeutic means for effecting
treatment practicing the instant invention.
Reference can also be made to any of numerous
authorities and references including, for example,
"Goodman & Gilman's, The Pharmaceutical Basis for
Therapeutics", (6th Ed., Goodman, et al., eds.,
MacMillan Publ. Co., New York, 1980).
The cochleates of the instant invention can be
used as a means to transfect cells with an efficacy
greater than using currently known delivery means,
such as liposomes. Hence, the polynucleotide
cochleates of the instant invention provide a
superior delivery means for the various avenue of
gene therapy, Mulligan, Science 260: 926-931
(1993). As Mulligan noted, the many possibilities
of treating disease by gene-based methods will be
enhanced by improved methods of gene delivery.
The cochleates of the instant invention also
serve as excellent means for delivering other
biologically relevant molecules to a host. Such
biologically relevant molecules include nutrients,
vitamins such as vitamins A, D, E or K, co-factors,
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enzymes, fatty acids such as polyunsaturated forms,
minerals including divalent cations such as
calcium, magnesium, zinc, iron or barium, flavors
and the like. Because the biologically relevant
molecule is contained within the cochleate, in a
non-aqueous environment, the biologically relevant
molecule essentially is stabilized and preserved.
As described hereinabove, the biologically relevant
molecule is added to the lipid solution and
processed to form a precipitated structure
comprising lipid and biologically relevant
molecule. As demonstrated herein, hydrophilic
molecules can be "cochleated", that is, can be made
part of the cochleate structure, with little
difficulty.
Also, suitable lipophilic biologically
relevant molecules, such as drugs and other
therapeutic compounds, are amenable to cochleation.
For example, lipophilic drugs such as eyclosporin,
ivermectin and amphoterioin are readily cochleated.
Other lipophilic drugs which are amenable to
incorporation into cochleates are acyclovir,
propanidid, propofol, alphadione, echinomycine,
miconazole nitrate, teniposide, didemnin B,
hexamethylmelamine, taxol, taxatere, melphalan,
adriamycin, 18-hydroxydeoxycorticosterone,
rapamycine, prednisolone, dexamethazone, cortisone,
hydrocortisone, pyroxicam, naproxen, diazepam,
verapamil, nifedipine.
The instant invention now will be described by
means of specific examples which are not meant to
limit the invention.
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Bovine brain phosphatidylserine in chloroform
was purchased from Avanti Polar Lipids, Birmingham,
Alabama in glass ampules and stored under nitrogen
at -20°C. Cholesterol (porcine liver) grade I,
a-D-octyl-glucopyranoside (OCG), fluorescein
isothiocyanate (FITC)-dextran (average mol. wt.
67,000), metriza~mide grade I, and cheaicals for
buffers and protein and phosphate determinations,
were obtained from Sigma Chemical Company, St.
Louis, Missouri. Organic solvents were purchased
from Fisher Scientific Co., Fairlawn, New Jersey.
Reagents for polyacrylamide gel electrophoresis
were from BioRad Laboratories, Richmond,
California. SloflO Sephacryl Superfine was obtained
from Pharmacies, Piscataway, New Jers~y. Thick
walled polycarbonate centrifuge tubes (10 ml
capacity) from Beckman Instruments, Palo Alto,
California, were used for vesicle preparations,
washes, and gradients. A bath type sonicator,
Model G112SPiG, from Laboratory Supplies Company,
Hicksville, New York was used for sonications.
Virus was grown and purified essentially as
describ~d by M.C. Hsu et al., Vjroloav, Vol. 95,
page 476 (1979). Sendai (parainfluenza type I) and
influenza (A/PR8/34) viruses were propagated in the
allantoic sac of 10 or 11 day old smbryonated
chicken eggs. Eggs were inoculated with 1-100 egg
infectious doses (103 to 105 viral particles as
determined by HA titer) in 0.1 ml of phosphate
buffered saline (0.2 gm/L KC1, 0.2 gm/L KH2P0', 8.0
gm/L NaCl, 1.14 gm/L NazFI-P04, 0.1 gm/L CaCl2, 0.1
gm/L MgC126H20 (pH 7.2)). Eggs were incubated at
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37°C for 48 to 72 hours, followed by incubation at
4°C for 24 to 48 hours. Allantoic fluid was
collected and clarified at 2,000 rpm for 20 minutes
at 5°C in a Damon IEC/PR-J centrifuge. The
supernatant was then centrifuged at 13,000 rpm for
60 minutes. This and all subsequent
centrifugations were performed in a Sorvall RC2-B
centrifuge at 5°C using a GG rotor. The pellets
were resuspended in phosphate buffered saline (pH
7.2) by vortexing and sonicating, followed by
centrifugation at 5,000 rpm for 20 minutes. The
pellet was resuspended by vortexing and sonicating,
diluting, and centrifuging again at 5,000 rpm for
20 minutes. The two 5,000 rpm supernatants were
combined and centrifuged at 13,000 rpm for 60
minutes. The resulting pellets were resuspended in
phosphate-buffered saline by vortexing and
sonicating, aliquoted, and stored at -70°C.
Sterile technique and materials were used
throughout viral inoculation, isolation, and
purification.
Virus stored at -70°C was thawed, transferred
to sterile thick-walled polycarbonate tubes and
diluted with buffer A (2 mM TES, 2 mM L-histidine,
100 mM NaCl (pH 7.4)). Virus was gelleted at
30,000 rpm for 1 hour at 5°C in a Beckman TY65
rotor. The supernatant was removed and the pellet
resuspended to a concentration of 2 mg viral
protein per ml of extraction buffer (EB) (2 M NaCl,
0.02 M sodium phosphate buffer (pH 7.4)) by
vortexing and sonicating. The nonionic detergent
a-D-octyl-glucopyranoside was then added to a
concentration of 2% (w/v). The suspension was
mixed, sonicated for 5 seconds and placed in a 37°C
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water bath for 45 minutes. At 15, 30 and 45 minute
incubation times, the suspension was removed
briefly for mixing and sonication. Nucleocapsids
were pelleted by centrifugation at 30,000 rpm for
45 minutes in a TY65 rotor. The resulting clear
supernatant was removed and used in the formation
of viral glycoprotein-containing cochleates. Some
modification of the above procedure may have to be
employed with other membrane proteins. Such
modifications are well known to those skilled in
the art.
A. DC Cochleat~s.
An amount of phosphatidylserine and
cholesterol (9:1 wt ratio) in extraction buffer and
non-ionic detergent as described hereinabove was
mixed with a pre-selected concentration of
polynucleotide and the solution was vortexed for 5
minutes. The clear, colorless solution which
resulted was dialyzed at room temperature against
three changes (minimum 4 hours per change) of
buffer A (2 mM TES N-Tris[hydroxymethyl]-methyl-2
aminoethane sulfc~nic acid, 2 mM L-histidine, 10o mM
NaCl, pH 7.4, also identified as TES buffer)
containing 3 mM CaCl2. The final dialysis routinely
used is 6 mM Ca2', although 3 mM Caz' is sufficient
and other concentrations may be compatible with
cochleate formation. The ratio of dialyzate to
buffer for each change was a minimum of 1:100. The
resulting white calcium-phospholipid precipitates
have been termed DC cochleates. When examined by
light microscopy (x 1000, phase contrast, oil), the
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suspension contains numerous particulate structures
up to several microns in diameter, as well as
needle-like structures.
B. LC Cochleates.
An amount of phosphatidylserine and
cholesterol (9:1 wt ratio) in extraction buffer and
non-ionic detergent as described hereinabove was
mixed with a pre-selected concentration of
polynucleotide and the solution was vortexed for 5
minutes. The solution first was dialyzed overnight
using a maximum ratio of 1:200 (v/v) of dialysate
to buffer A without divalent cations, followed by
three additional changes of buffer leading to the
formation of small protein lipid vesicles. The
vesicles were converted to a cochleate precipitate,
either by the direct addition of Caz' ions, or by
dialysis against two changes of buffer A containing
3 mM Ca2+ ions, followed by one containing buffer A
with 6 mM Ca2+.
IM1LONE RBB~O;~BB TO OI~tAL~Y
DEL~CVERRD PROT:~,;~~COC LB~lIT~ VACCI118B
To make the vaccine, influenza virus was
grown, purified, and the glycoproteins and lipids
extracted and isolated as described in Example 1.
Protein-cochleates were made according to the '~LC
cochleate~' procedure described above.
Cochleate vaccines containing the
glycoproteins and lipids from the envelope of
influenza virus and phosphatidylserine and
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cholesterol were given to mice by gradually
dispensing 0.1 ml liquid into the mouth and
allowing it to be comfortably swallowed. Figures
4(A) (from Experiment A) and 4(B) (from Experiment
B) show resulting total circulating antibody levels
specific for influenza glycoproteins, as determined
by ELISA. Antibody titer is defined as the highest
dilution that still gives the optimal density of
the negative control.
In Experiment A that generated the data shown
in Figure 4(A), initial vaccine doses of 50, 25,
12.5 or 6.25 ~cg of glycoproteins (groups 1 through
4 respectively) were administered at 0 and 3 weeks.
The third and fourth immunizations (6 and 19 weeks)
were at one fourth the dose used for the initial
two immunizations. Bleed 1 - Bleed 6 occurred at
0, 3, 6, 9, 19, and 21 weeks. The data demonstrate
that high circulating antibody titers can be
achieved by simply drinking cochleate vaccines
containing viral glycoproteins. The response is
boostable, increasing with repeated administration,
and is directly related to the amount of
glycoprotein in the vaccine.
Those observations were confirmed and extended
in Experiment B that generated the data shown in
Figure 4(B). The dose range was expanded to
include 100 dug and 3.1 ~g initial doses. Vaccine
was given at 0, 3 and 15 weeks, with the third
immunization at one fourth the dose of the initial
two. Bleed 1 to Bleed 6 occurred at 0, 3, 6, 15
and 16 weeks. Circulating influenza
glycoprotein-specific responses were detectable
after a single administration for the top five
doses, and for all groups after two feedings. The
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data shown is for pooled sera from each group, but
all mice given the four highest doses, and four of
five mice in groups f ive and s ix, responded to the
vaccine with circulating antibody titers ranging
from 100 to 102,400. Group seven, which received
no vaccine, had titers less than 50 for ml! mice at
all time points.
The antibody response is long lived. Titers
13 weeks after the third immunization (Figure 4(A),
bleed 5) and 12 weeks after the second immunization
(Figure 4(B), bleed 4) remained the same or within
one dilution higher or lower than seen at 3 weeks
after the previous boost.
To determine whether oral administration of
the subunit vaccine described in Example 2 could
lead to protective immunity in the respiratory
tract, the mice described in Experiment B of
Example 2 were immunized with cochleates at 0, 3
and 15 weeks. The immunized mice were challenged
by intranasal application of 2.5 x 109 particles of
influenza virus at 16 weeks. Three days after
viral challenge, mice were sacrificed, and lungs
and trachea were obtained. The entire lung or
trachea was triturated and sonicated, and aliquots
were injected into embryonated chicken eggs to
allow amplification of any virus present. After
three days at 37°C, allantoic fluid was obtained
from individual eggs and hemagglutination (HA)
titers were performed.
Mice were also challenged with live influenza
intranasally following oral cochleate
administration in Experiment A of Example 2. Lungs
were obtained three days later and cultured to
detect presence of virus.
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The combined data for the two experiments is
given in Table 1. The results also are shown
graphically in Figure 5.
Vaccine Tracheal Lung~Z Lunga9
Doee
J~9 Infected/TotInfected/TotInfected/Tota
Protein al al 1
100 0/5 0/5 0/5
50 2/5 0/5 2/10
25 0/5 0/5 1/10
125 1/5 0/5 1/10
6.25 0/5 5/5 6/10
3.12 4/5 5/5 5/5
0 5/5 5/5 9/10
I
1. Mice from Experiment B.
2. Mice from Experiment B.
3. Mice from Experiments A and B.
The data in Table 1 shows that all five of the
unvaccinated mice had sufficient virus in the trachea to infect
the embryonated chicken eggs (greater than 103 particles per
trachea or at least one egg infectious dose {EID) per 0.1 ml of
suspension). In contrast, the oral vaccine provided a high
degree of protection from viral replication in the trachea. All
mice in groups 1, 3 and 5 of Experiment B were negative for
virus. Two mice in group 2, 1 in group 4, and 4 in group 6 (the
lowest vaccine dose) of Experiment B had sufficient virus to
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test positive in this very sensitive assay used to detect
presence of virus.
The oral protein cochleate vaccine also provided
protection against viral replication in the lungs. All twenty
mice which received the four highest doses of vaccine were
negative for virus when lung suspensions were cultured in
embryonated chicken eggs (Table 1). All mice in the groups
immunized with 6.25 beg and 3.1 ~cg glycoproteins and all mice in
the unvaccinated control were positive for virus.
Even in the lowest two vaccine doses, there was some
inhibition of viral replication. When lung suspensions were
diluted 1/10 and inoculated into eggs, only one animal in the
groups immunized with 6.25 ~cg was positive, as compared to three
in the groups immunized with 3.12 ~g and three in the
unvaccinated control. Culturing of 1/100 dilutions resulted in
one positive animal in each of the groups immunized with 6.25
and 3.12 fig, but 3 of 5 remained positive in the unvaccinated
group. In addition, for the two animals in the group that was
immunized with 3.12 ~cg, but Which were negative at 1/100, only
50% of the eggs were infected at 1/10 and had low HA titers. In
contrast, for the unvaccinated group, all eggs were infected and
produced maximal amounts of virus at 1/10 and 1/100 dilutions.
C57BL/6 mice were given cochleates containing Sendai
virus glycoproteins orally at 0 and 3 weeks. They were bled at
0 (bleed 1), 3 (bleed 2}, and 6 (bleed 3) weeks. Group 1
received approximately 50 ~cg protein, Group 2 about 25 ~cg, Group
3 about 12.5 fig, Group 4 about 6.25 fig, and Group 5 (negative
control) received 0 ~g protein. The levels of Sendai specific
antibodies in the serum pooled from 5 mice in each dose group
were determined by ELISA. The results are shown in Figure 6.
It can be seen that strong antibody responses were generated,
that the magnitude of the response was directly related to the
immunizing dose, and that the magnitude of the response
increased (boosted) after a second immunization.
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The response was extremely long-lived. The response is
predominantly IgG, indicative of the involvement in T cell help
and establishment of long-term memory cells associated with a
secondary immune response. Surprisingly, the lowest dose which
initially had the loawest response, now had the highest
circulating antibody levels. This may be due to the immune
system's down regulation of the very high responses originally
but allowing the low response to slowly climb. This may also
indicate a persistence and slow release of antigen. It is also
interesting and consistent with the use of the oral route of
immunization that significant IgA titers are generated and
maintained.
A 50 ~g protein dose of Sendai glycoprotein-containing
cochleates was given orally. Two weeks later the animal (BALB/c
mouse) was sacrificed and spleen cells obtained. Cytolytic
activity of the spleen cells was measured by their ability to
cause the release of chromium-51 from target cells presenting
Sendai antigens. The non-immunized mouse did not kill Sendai
virus (SV) pulsed cells with in culture restimulation (N/SV/SV)
or non-Sendai presenting cells (N/N/N). (Figure 7) In contrast,
Sendai cochleate immunized mice killed SV pulsed targets to a
very high degree and rion-pulsed targets to a lesser degree.
Cytolytic activity is crucial to clearance of cells infected
viruses, or intracellular parasites or to cancer cells. It is a
highly desirable activity for a vaccine to induce, but
classically has not been seen with most non-living vaccines.
This is an important feature of protein-cochleate vaccines.
Eight week old BALB/c female mice were immunized IM
twice with various polynucleotide-cochleate formulations,
polynucleotide alone and controls and then splenocytes from the
mice were tested for the ability to proliferate in response to a
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protein encoded by the polynucleotide.
Cochleates with and without fusogenic Sendai virus
protein were prepared as described hereinabove. The
polynucleotide used was the pCMVHIVLenv plasmid. The solution
containing lipid and extracted Sendai virus envelop proteins as
described hereinabove and polynucleotide were mixed at a 10:1
(w/w) ratio and 50:1 (w/w) ratio. That protocol yielded four
groups, cochleate/DNA, 10:1; cochleate/DNA, 50:1;
SV-cochleate/DNA, 10:1; and SV-cochleate/DNA, 50:1. Naked DNA
was used at a rate of 10 ~g/mouse and 50 ~g/mouse. The control
was buffer alone. Mice were immunized twice, 15 days apart at
50 ~1/mouse.
Splenocytes were obtained and tested in a T-cell
proliferation assay using tritiated thymidine, as known in the
art. Control cultures contained no antigen or con A. The
antigen used was p18 peptide, at 1 mM, 3 mM and 6 mM. Cells
were harvested at days 2, 4 and 6 following preparation of the
splenocyte cultures.
The naked DNA provided a marginal response above
background. All four cochleate preparations yielded a
p18-specific response which increased over time. At six days,
the response was about four times above background.
The DNA concentration range at the 10:1 ratio was about
120-170 ;eg/ml. At the 50:1 (w/w) ratio; the DNA concentration
was about 25-35 ~g/ml.
The polynucleotide-cochleates were exposed to
micrococcal nuclease and little or no nucleic acid degradation
was observed.
The polynucleotide encapsulation efficiency was found to
be about 50% based on quantificat ~n of free DNA from lipid,
that is present in the supernatar- following a precipitation
reaction. After washing the precipitate and opening the
structures by removing cation about 35% of the DNA was
recovered.
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EX~MBhE 5
In similar fashion, splenocytes from animals immunized
as described in Example 4, were tested for antigen specific
cytotoxic activity using a chromium release assay using labelled
H-2 compatible target cells known to express an HIV protein,
such as gp160. The responder cells can be stimulated by brief
exposure to purified HIV peptides.
On prestimulation, animals exposed to polynucleotide
cochleates demonstrated specific cytotoxic splenocytes directed
to gpl6o, with nearly 100% cytotoxicity observed at an
effector:target ratio of 100.
Fifteen mg of insulin were added to 15 ml of extraction
buffer (EB) in a 50 ml plastic tube. Then 300 mg of OCG were
added to the mixture. The resulting suspension was colloidal
and not clear at pH '~.4. The solution was titrated with 1 N
NaOH to pH 8.5, resulting in a clear solution.
In a separate vessel, 6.8 ml of a 10 mg/ml solution of
phosphatidylserine and 1.5 ml of a 5 mg/ml solution of
cholesterol were mixed amd then dried to yield a thin film. The
insulin solution was added to the vessel yielding a colloidal
suspension. The suspension was vortexed for seven minutes and
then set on ice for one hour. The pH of the solution was
adjusted to 9-9.5 with 1 N NaOH, the sample was filter
sterilized and placed in dialysis tubing at about 2 ml per bag.
Two different dialysis schedules were used.
A. DC cochleates:
1. +z 100Zm1 overnight 1 x TES pH 9.0 containing+2 3
mM +Ca , Zn or Mg
2. +Z 2502 ml 4h 1 x TES pH 8.5 containing+Z 3
mM Ca , Zn or Mg
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3, 2502m1 4h 1 x TES pH 8.0 containing 3
+
+2 Zn or Mg
Ca ,
mM
2502m1 4h lxTES pH 7.4 containing 6
;
;Z Zn or ~ Mg
Ca ,
mM
~g, LC cochleates:
1, 100 overnight x TES, pH 9.0
ml 1
2, 250 4h, 1 x TES, pH 9.0
ml
3, 250 4h 1 x TES, pH 9.0
ml
4, 1002m1 overnight x TES, pH 9.0 containing 3
; 1
;
;
+Z Zn or Mg
Ca ,
mM
5, ' 250 4h 1 x TES, pH 8.5 containing
ml 'z
'
2
or Mg'
3 mM Ca
, Zni
6, 250 4h 1 x TES, pH 7.4 contair~ing
ml +z
+
Z
or Mg
6 mM Ca
, Zn'
Following dialysis, the resulting precipitate was found
to comprise numerous cochleates.
EgAMPLE 7
Mice were given insulin cochleate samples orally. Serum
glucose levels were measured at 0 time, (prior to cochleate
administration), 30 min. and 60 min. post administration using
standard methods. Cochleate formulations of Example 6 with a
starting concentration of 1 mg insulin/ml solution were used.
Each mouse was administered 100 u1 or 200 ul.of the designated
preparations as indicated: For comparison, one mouse was given
the standard commercial human insulin, Humulin R, by
intraperitoneal administration.
* Trademark
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Sample Volume Given Serum Glucose mg/dl
0 Time 30 min. 60 min.
LC Ca++ 200 u1 100 49.12 43
I~C Ca++ 200 u1 102.9 252.4 61.9
.
Humulin R 200 u1 88.8 66 48.5
*
Oral administration of insulin affected serum
glucose levels. .
EB,AMPLE 8
Insulin cochleates as produced in Example
. were fed orally to three-month-old female BALB/c
mice made diabetic through intraperitoneal
injection of streptozotocin, practicing known
methods. Two days after exposure to
streptozotocin, the mice were allocated into groups
of five and administered with oral insulin
cochleates at.200 ~1 per mouse. Other mice were
injected with 2 IU of Fiumulin R.
Serum samples were'obtained at time 0, prior.
to insulin dosing, and two hours post insulin
administration. Glucose levels were measured using
a kit from Sigma (St. houis). Control animals were
untreated, that is, received no streptozotocin or
* Trademark
SUBSTITUTE SI~IEET (RULE 26)
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- 36 -
Figure 8. orally administered insulin, simply by
drinking, was effective in reducing blood glucose
levels. No reduction in blood glucose was observed
in control animals.
While the invention has been described in
detail and with reference to specific embodiments
thereof, it will be apparent to one skilled in the
art that various changes and modifications can be
made therein without departing from the spirit and
scope thereof.
NOTRIENT-COCHLEATES
ERAMPLE 8
Vitamin A in cochleates
Vitamin A (retinol) is sensitive to air-
oxidation and is inactivated by ultraviolet light.
Stability of vitamin A is enhanced by its
encapsulation into the intra-bilayers of
cochleates. Incorporation of vitamin A into the
intra-bilayer phospholipid region of a cochleate
was achieved as follows: appropriate proportions of
vitamin A, phosphatidylserine and cholesterol were
dissolved in an organic solvent such as chloroform
or a 1:1 methanol: chloroform mixture. The solvent
was then removed under reduced pressure to yield a
lipid-vitamin film. Buffer was added and the
mixture was vortexed for several minutes. The
resultant dispersion was then dialyzed at room
temperature as in example 2.A against three changes
of buffer A containing 3 mM CaCl2. Vitamin A-
cochleates were obtained as a precipitate.
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~E11~IPLE 4
Bol~runaaty~at.d fatty a~~aids is coahleatee
Unsaturated fatty acids are biologically
important in that they control the level of
cholesterol in blood and are the precursors of
prostaglandins. The limitation in incorporating
polyunsaturated fats in food is their
susceptibility to oxidation. In the presence of
oxygen, unsaturated fatty acids undergo a series of
reactions called autoxidation, whose final products
are aldehydes and ketones, which provide fishy
unpleasant odor and flavor. An interesting way to
control autoxidation of unsaturated fats is to
incorporate them into the bilayers of a cochleate.
The polyunsaturated fatty acids (PUFA) will be
placed in close contact with oxygen-stable
saturated fatty esters of the phosphatide.
Incorporation, for example, of fish oils (which are
rich in PUFA) into the intra-bilayer phospholipid
region of a cochleate was achieved as follows:
appropriate proportions of fish oil,
phosphatidylser~.ne and cholesterol (or optionally
alpha-tocopherol as a stablizer and autoxidant),
were dissolved in organic solvent such as
chloroform or a 1:1 methanol: chloroform mixture.
The solvent was then removed under reduced pressure
to yield a lipid film. Buffer was added and the
mixture was vbrtexed for several minutes. The
resultant dispersion was then dialyzed at room
temperature as in example 2.A against three changes
of buffer A containing 3 mM CaCl2. PUFA-cochleates
were obtained as a precipitate.
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B~~~PhE 10
Flavors are volatile and sensitive to
oxidation. Controlled release and enhanced physical
and chemical stability can be achieved by the
encapsulation of flavors into cochleates.
Incorporation of a flavor based on cinnamon oil
into the intra-bilayer phospholipid region of a
cochleate can be achieved as follows:
phosphatidylserine and cholesterol were dissolved
in an organic solvent such as chloroform or a 1:i
methanol: chloroform mixture, and an appropriate
proportion of cinnamon oil dissolved in ethanol was
added. The solvent was then removed under reduced
pressure to yield a film. Huffer was added and the
mixture was vortexed for several minutes. The
resultant dispersion was then dialyzed at room
temperature as in example 2.A against three changes
of buffer A containing 3 mM CaClz. Cinnamon oil-
cochleates were obtained as a precipitate.
hjPOPBILIC DROG COC8L8~1T$I~
~a~L$ ii
llcyclo~rir in aoch~,oat~s
Incorporation of acyclovir into the intra-
bilayer phospholipid region of a cochleate can be
achieved as follows: acyclovir/phosphatidylserine
in an appropriate drug to lipid ratio was dissolved
in an organic solvent such as chloroform or a 1:1
methanol: chloroform mixture. The solvent was then
removed under reduced pressure to yield a
homogenous film. Buffer was added and the mixture
was vortexed for several minutes at a temperature
above the transition temperature of the lipid. The
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excess drug, if any, was separated from the
liposome containing acyclovir by repeated washing
with PBS and centrifugation, the supernatant was
discarded, and the pellet resuspended in PBS. The
liposome suspension was then dialyzed at room
temperature as in example 2.A against three changes
of buffer A containing 3 mM CaClZ. Acyclovir-
cochleates were obtained as a precipitate.
~~~L~ iz
8y~~ oaort,~sons ~y c~,g9~hll~ata
Incorporation of hydrocortisone into the
intra-bilayer phospholipid region of a cochleate
c a n b a a c h i a v a d a s f o 1 1 o w s
hydrocortisone/phosphatidylserine in an appropriate
drug to lipid ratio were dissolved in an organic
solvent such as chloroform or a 2:1
methanol: chloroform mixture. The solvent was then
removed under reduced pressure to yield a
homogeneous film. Buffer was added and the mixture
was vortexed for several minutes at a temperature
above the transition temperature of the lipid. The
excess drug, if any, was separated from the
liposome containing hydrocortisone by repeated
washing with PBS and centrifugation, the
supernatant was discarded, and the pellet
resuspended in PBS. The liposome suspension was
then dialyzed at room temperature as in example 2.A
against three changes of buffer A containing 3 mM
CaCl2. Hydrocortisone-cochleates were obtained as a
precipitate.
