Note: Descriptions are shown in the official language in which they were submitted.
CA 02318556 2000-07-14
AttorWSy Edacltet No. O~~Q4Q31p1.9R
Lawrertcae T. Boni
Micba~el M. 8ac~jany
i~ary Neviile
Richard J. Robb
Mircea Popescu
L~IPOMATP~PARA~ON
BACKG80iJ'~ OF'i'i3E INV~N'I~ON
Ligosomes are in>~iagly i~ottarn as vehicles for the delivery of
pbarmaceutieal agents. Iu addition w such applicacioas, their nse as
iuununologic aQjuvaurs is
especially important.
TSradioonal vaccines typically involve immuniza~cia~ with either purif>s;d
antigen
or an atxrauaced pathogen. These aac~iooal methods suffer, for example, fro~a
the danger of
atonally iufecti~ag People while g to iuununize them. Another persistent
problem
with purifrsd antigens is that they do not always i~tce a long-term iuunutns
respo~e, toad
sornetim~es induct no ~respoasr at sll. It has boen discovered, hawevrr, that,
while direct
immunization with ccrtaitt antigens alone coo generate a short-terra itnuu,ar
response,
i~uizatian with antigen entrapped is liposomes can induce a long-term mpoase
which is
essential far any effet~tive vaccine. 'Thus, Iiposames offer promise in
overcoming obstacles too
txaditional i:mmuaizatimn-
In a typical process for the m~at~Facau'e of liposoxnes camposod of more than
otae
Iipid or lipid and lipophilic molocule, the components are dissolved is an
organic solvent.
Next, one of two general procedures u~c followed. See, e.g., 8angham, Cbem.
ghys. Lipids
X4:275-2BS (I993); Szal~a et al., proc. Natl. Aced. Sa. ?5:4194-98 (1978); and
Kita er al.,
BiQCltim. Biophys. Act$ 728:359-48 (1983). f~rher tnetbods are found in
WtJ192111842,
W0195139121, GB 2.157,172, WOI9SI045Z3, apd W0197129769.
2D ~ In the fu'st approach, tlu lipid rnbaure in as organic solvent is dried
to
a thin film acing a rotovap. An aqueous phase, usually containing a salutr to
be rnc~apsularad,
is then added with vortexi,ng co form liposomes. is the other aggroach an
aqueous piaa~se, with
solute to be eatr~ppOd, ig added to the organic phase wisich is su>uoqueutty
retaoved by eithsr
vas, or sparging with au inert gas, thus forusing liposomes.
A':~'~.'~'~c~J ~'~Ef
:.297327.1
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WO 99/36056 PCTNS99/00924
The known methods, however, have significant technical, economic and
environmental drawbacks. Specifically, the thin film method can not be readily
scaled up.
Moreover, in the mixed organic/aqueous phase systems, the removal of the
organic phase is
cumbersome and often incomplete. Thus, the final product will contain residual
organic
solvent, which is potentially toxic and carcinogenic. Indeed, both methods
typically employ
such toxic substances, e.g., chloroform, acetonitrile and acetone.
Accordingly, improved methods for the manufacture of liposomes are needed
which avoid these failings. Moreover, a need exists for improved liposome
compositions for
use in biomedical applications that can be manufactured by these improved
processes, yet are
highly effective as immunological and pharmaceutical mediators.
SUM1VIARY OF THE INVENTION
It is, therefore, an object of the invention to provide superior liposome-
based
vaccine compositions. According to this object of the invention, a Lipomatrix
composition is
provided, which forms liposomes only upon rehydration. This composition is
particularly
suited to the manufacture of novel tumor vaccines that induce superior immune
responses.
Other objects of the invention include providing a process of Lipomatrix
preparation which (a) can be scaled up, (b) will not contain unacceptable
residual solvents and
(c) is quick and simple from a manufacturing standpoint. Further to these and
other objects,
methods are provided for economically and safely producing a Lipomatrix, which
can be used
to make liposomes that are suitable for a wide variety of biomedical uses, and
especially for
vaccine applications.
In one embodiment, a method of preparing a Lipomatrix is provided where a
water miscible organic phase, containing a phospholipid and at least one other
lipid is mixed
with an aqueous phase in a ratio of from about 100:1 to about 5:1 (v/v), then
drying the
mixture.
Another embodiment of the invention provides a Lipomatrix containing
between about 20 Mol % and about 60 Mol % cholesterol.
In yet another embodiment, a method of preparing liposomes is provided wherein
the inventive Lipomatrix is rehydrated.
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WO 99/36056 PCT/US99/00924
In still another embodiment, a vaccine is provided which comprises liposomes
prepared from a Lipomatrix according to the methods of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic illustrating the general process for preparing a
Lipomatrix
according to the invention.
Figure 2 shows the fluorescence emission spectra of carboxyfluorescein
entrapped in
a Lipomatrix compared to what would be expected for 100% entrapment.
Figures 3A and 3B are electron micrographs. Figure 3A shows the absence of
liposomal structures in a typical Lipomatrix before lyophilization and Figure
3B shows the
formation of liposomes after hydration of the Lipomatrix.
Figures 4A and 4B show interferon gamma (IFN-y) production of mouse lymph
node cells (Figure 4A) and spleen cells (Figure 4B) in response to antigen
challenge. The
mice were immunized with liposomal MUC-1 prepared by hydrating the Lipomatrix
formulations described in Example 3.
Figure 5 shows interferon gamma (IFN-y) production of mouse lymph node cells
and spleen cells in response to antigen challenge. The mice were immunized
with a
liposomal MUC-1 vaccine prepared by hydrating Lipomatrix formulations
containing
varying amounts of cholesterol as described in Example 4.
Figure 6 compares the differential scanning calorimetry (DSC) heating scans of
liposomal MUC-1 preparations made by hydrating a Lipomatrix formulated at 50
Mol %
cholesterol and that of DPPC liposomes at the same bulk lipid concentration
(20 mg/mL).
Figure 7 shows the Raman vibrational spectroscopic profile of a Lipomatrix
formulation prepared as described in Example 1. In panel A, solid and dotted
lines
represent two different sites in the lyophilized film. Panel B shows a Raman
profile for the
hydrated formulation.
Figure 8 shows the interferon gamma (IFN-~y) production by lymph node cells
and
spleen cells in response to antigen challenge after immunization with a
liposomal MUC-1
vaccine prepared from a Lipomatrix. Time points represent the hours that the
hydrated
Lipomatrix formulation stood at room temperature before subcutaneous injection
into mice.
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WO 99/3b056 PCT/US99/00924
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a Lipomatrix, which is essentially comprised of lipid
lattices (or stacked bilayers), which forms liposomes upon hydration. The
invention also
provides a simplified method for producing highly effective liposome
preparations, by
hydration of a Lipomatrix. This method of producing liposomes from a
Lipomatrix
overcomes many of the above-described obstacles to the efficient and safe
manufacture of
liposomes. In a specific example, the usefulness of the Lipomatrix is
demonstrated in the
manufacture of a mucin-based cancer vaccine.
The inventive Lipomatrix is essentially a dried (e. g. , lyophilized)
composition of
lipid that is capable of forming liposomes upon reconstitution. In this dried
state, the
composition is characterized as a matrix of stacked bilayers, and is
essentially liposome-free.
It is comprised in its dried form of mostly lipid, with at most trace amounts
of solvent and less
than about 5 percent water by weight. Trace amounts of solvent are generally
less than about
0.1 % and typically less than about 0.05 % by weight. Some exemplary
compositions contain
(by weight) about 93-94 % lipid and some contain less than about 3-4 % water.
Prior to drying, the Lipomatrix exists as an essentially liposome-free
suspension
of lipid. In this state, the Lipomatrix is comprised mostly of solvent and
water, with lipid
levels usually being less than about 10 percent, by weight. In many cases,
however, the lipid
will be present at even lower levels, such as less than about five percent by
weight. Some
exemplary compositions have lipid at levels less than about 1.5 % by weight.
An embodiment
below utilizes lipid levels of about 0.9 % . Solvent levels will usually be
kept above about 80 %
and in some instances may be about 95 % . Some exemplary compositions have
between about
85 % and about 90 % , while others will be above about 90 % . On the other
hand, water is
usually present at somewhat lower levels, typically less than about 20 % .
Many compositions
have between about 10 % and about 15 % water by weight.
In suspension (prior to drying), some of the Lipomatrix compositions have
solvent:water ratios of from about 5:1 to about 20:1 (vol./vol.); two
exemplary compositions
have ratios of 7:1 and 9:1. Especially where vaccine applications are
contemplated, the
lipidaolvent mass ratio should be above about 1:20, and it usually will be
less than about
1:50. The higher end of this range (e.g., from about 1: 35 to 1: 50) is
preferred for its ability
to produce superior vaccines.
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Briefly, the basic method involves first creating an organic phase, by
dissolving
appropriate lipids in a water miscible organic solvent, which is mixed with a
small volume of
an aqueous phase to induce molecular ordering, i. e. , formation of the
Lipomatrix. An
optional sterilizing filtration step is included either before or after this
mixing. The resulting
Lipomatrix is lyophilized or freeze-dried. The dried mixture can be hydrated
in an
appropriate medium, thereby spontaneously forming liposomes capable of
entrapping an
aqueous solute. In contrast to the prior art, liposomes are not appreciably
formed in the
present method at any time prior to hydration. Indeed, as demonstrated below
in ~ the
Examples, no liposomal structures are detected prior to hydration. Thus,
unlike prior art
methods, the instant methods do not involve pre-forming liposomes that are
dried and merely
rehydrated by the addition of water. In fact the Lipomatrix preparation is
essentially
liposome-free until it is hydrated, as set out below.
Notably, the instant methods can be used to prepare a Lipomatrix, which can be
hydrated to make highly effective liposome compositions that have a high
cholesterol content.
1 S It is believed that the added cholesterol broadens the transition
temperature and eliminates
domains of either phospholipid, cholesterol or other lipophilic components and
their
combinations. See Ladbroke et al., Biochim. Biophys. Acta 150:333-40 (1968).
These
previous studies employed cholesterol in liposomes to decrease leakage of
solute from the
liposome, for modifying liposome size or for mimicking plasma membrane
compositions.
The art did not recognize, however, that certain liposomes containing high
cholesterol content
have superior adjuvant properties. As demonstrated below in the Examples, when
such
liposomes are prepared from the inventive Lipomatrix, they have surprisingly
better immuno-
stimulatory properties.
2S Methods for Producing a Lipomatrix
The present methods for preparing Lipomatrix involve, first, preparing an
organic
phase by dissolving at least one lipid in a water-miscible organic solvent.
Suitable lipids
include phospholipids, in particular lecithins, phosphatidylglycerols,
phosphatidylethanolamines, phosphatidylserines and other natural and synthetic
compounds
known in the art. See, for example, WO 91 /04019 ( 1991 ) at pages 8 and 9.
Preferred
phospholipids specifically include dimyristoyl phosphatidylcholine (DMPC),
dipalmitoyl
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WO 99/36056 PCT/US99/00924
phosphatidylcholine (DPPC) and dimyristoyl phosphatidylglycerol (DMPG). Other
suitable
lipids include sterols, and especially cholesterol. Also suitable are
glycolipids and lipid
adjuvants, such as monophosphoryl Lipid A (MPL) or Lipid A.
In a preferred Lipomatrix suspension, the final concentration of phospholipids
is
between 15 mg/mL and about 36 mglmL, depending of course on the solubility of
the
particular lipids) used in the solvent chosen. Mild heating, for example
between 50 °C to
about 60 °C, may also be employed in the Lipomatrix formation. The
degree of heating
optionally used will depend in large part on the solubility and stability of
the various organic
phase components.
The inventive Lipomatrix is particularly useful when manufactured with high
cholesterol content. For example, the inventive methods can include in the
phospholipid
matrix about 20 Mol % or more of cholesterol. Some preferred Lipomatrix
formulations
contain from about 30 Mol % to about 60 Mol % cholesterol. The organic solvent
should be
water miscible and is usually an aliphatic alcohol. Preferred aliphatic
alcohols include, but are
not limited to, ethanol and tent-butanol. Other organic solvents, and
especially other alcohols,
may be employed. They should, however, be amenable to drying by, for example,
lyophilization, and they should be non-toxic. Thus, solvents such as those
typically employed
in the thin film method are generally unacceptable.
Second, an aqueous phase is provided. The aqueous phase may contain one or
more buffers, salts, and bulking agents. Preferred bulking agents include
sugars, and
preferred sugars include mannitol. Appropriate buffers, salts and bulking
agents preferably
are physiologically compatible and are widely known to those in the art. Of
course, the
concentration of these buffers, salts and bulking agents chosen will depend
primarily on
physiological compatibility, will be understood by those in the art.
Third, the organic phase and the aqueous phase are either mixed and optionally
sterile filtered, or optionally sterile filtered separately and then mixed.
When mixing, the
ratio of organic phase to aqueous phase is preferably from about 7:1 to about
9:1
volume/volume. This ratio, however, could be as high as about 100:1 and as low
as about 5:1
organic:aqueous. Ranges such as from about 20:1 to about 6:1 are also
acceptable. In
addition the lipidaolvent mass ratio should be between 1:20 and about 1:50,
with the higher
end of that range being preferred (e.g., from about 1:35 to 1:50). The mixing
can be done at
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CA 02318556 2000-07-14
Attorney DoClCet No. 404031019$
ambient txmpcratures, but may Ix done at temperatures as high as the highest
inching
temperacurc of the lipids employed. At this stage, although molecular ordcting
occurs and
open bilayers are forged (i. e. , Lipomauix), liposotnes arc not detectable.
' The Lipomatrix can he dried by lyophilization or other suitable means. The
Lipomatrix may be dried in bulk, prior to drying, it can be divided inm
aliquots of a suitable
size. Typically, the Lipomatru solution is aliquotcd into vials with
continuous mixing,
followed by lyophilization. The resulting Lipotnatrix #ormulatiou is stable
and suitable for
storage.
The formation of liposoomes is a~otaplisbed when the dried Ligomauix film or
cake is hydrated with a scalable aqueous solvent, such as water. saline, or an
appropriate
buffer, optionally containing a sohue far encapsuluion. The tempt-iaturc of
the hydration
solution znay be ambient to above tha trans~idon teuiperature of the highest
melting lipid. Only
upon hydration, are liposames formed which arc capable of entrapping an
aqueous solute.
This is a significant siznplifxcatian over the art, which relies on pre-
forming liposonzes prior to
drying. Moreover, because the prior art methods relird oa such pre-formation,
stabilizers
were needed to maintain tlse integrity of the tiposoa3as. Such stabilisers are
unnecessary in
the instant methods because liposotnrs are not formed grior to hydration.
Uses of the ~.ipoma~x
A Lipomatrix prepared acco=ding to the invention can be used in a wide
variety of applications after hydrating to form liposotnes, esprciaIly
biomedical
applications. For example, they cart be usrd to deliver a wide varirty of
pharuza,cologically
active agents. Thus, lipophific agents, for exarctplc hydrophobic peptides,
may be iaeludcd
in the organic phase. In addition, hydrophilic pharmacologically arrive agents
may be
entrapped within the resultant lipasom~es upon hydration. Charged molecules
might be
eleetrostatieally associated with the phnspholipids of thr liposomcs. Examples
of suitable
lipophilic and hydrophilic pharmacological agents can be found in Papescu et
al., 1J.S.
Patent 110. 5, I45,93fJ (1992), which is hereby incorporated by reference.
Other
pharmacologically activr agents include, for example, adjutants, cytokines,
antibodies and
~Y off' ~w'n pharmaceuticals. Especially useful pharmacological agents ir~lude
cyto>'iines, such as interleukin-2 (1L-'~), grauulcxyte-macrophage colony
stimulating factor
(GM-CSF) and iatrrtcron-garztn~a (IFN-y), which tray
A~;~r~~ ~c~ .Sl~~'
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WO 99/36056 PCT/US99/00924
be used alone or in conjunction with other agents. Combinations of any of
these agents are
also envisioned.
The inventive methods are especially useful in the manufacture of vaccines.
Moreover, nearly any type of antigen, but especially tumor antigens, may be
used. Tumor
antigens may be derived, for example, from lung cancer, colon cancer,
melanoma,
neuroblastoma, breast cancer, ovarian cancer and the like. A preferred tumor
antigen is
MUC-1 and related antigenic peptides. MUC-1 mucin is a high molecular weight
glycoprotein with a protein core consisting of tandem repeats of a 20 amino
acid sequence and
highly-branched carbohydrate side chains. Many human adenocarcinomas, such as
breast,
colon, lung, ovarian and pancreatic cancers, abundantly over-express and
secrete
underglycosylated MUC-1 protein. Importantly, a high level of MUC-1 mucin
expression is
associated with high metastatic potential and poor prognosis. MUC-1 is,
therefore, a
clinically significant marker for these cancers. Particularly useful antigenic
MUC-1 peptide
derivatives are based on the 20 amino acid repeat sequence.
In addition to tumor antigens, other clinically relevant antigens include
allergens,
viral antigens, bacterial antigens and antigens derived from parasites.
Antigens are usually
macromolecules such as peptides, lipids, carbohydrates and combinations
thereof which may
simply be mixed together or covalently linked, as in glycopeptides,
glycolipids.
Typical vaccine compositions comprise liposomes hydrated from the inventive
Lipomatrix formulations containing an antigen, such as a tumor antigen.
Additionally,
vaccine compositions may contain one or more immunomodulators. An
immunomodulator is
any substance that alters the immune response, and preferably stimulates the
antigenic immune
response. Typical immunomodulators include adjuvants, such as monophosphoryl
Lipid A
and Lipid A. Other immunomodulators include lymphokines and cytokines, and
specifically
interleukins, in particular IL-2.
The vaccines are typically formulated using a pharmaceutically acceptable
excipient. Such excipients are well known in the art, but typically will be a
physiologically
tolerable aqueous solution. Physiologically tolerable solutions are those
which are essentially
non-toxic. Preferred excipients will either be inert or enhancing with respect
to antigenic
activity.
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In the Examples below, an anti-tumor vaccine is prepared which comprises
liposomes containing a synthetic MUC-1 peptide, a tumor antigen, and using
monophosphoryl
Lipid A (MPL) or Lipid A as an immunomodulatory adjuvant. See Koganty et al. ,
DDT 1:
190-98 (1996); Alving et al., In Liposomes and Immunology, pp. 67-78 (1980).
The MUC-1
peptide is a synthetic peptide version with antigenic properties similar to
the parent MUC-1
glycoprotein. A vaccine formulated with this peptide antigen is currently
under clinical
investigation. See Koganty et al. DDT 1:190-198. Various formulations were
tested in mice
for the induction of an antigen-specific T cell response.
EXAMPLES
Example 1:
Lipomatrix Formation. The appropriate stock reagents in ethanol of DPPC (200
mg/mL),
cholesterol (50 mg/mL), MUC-1 lipopeptide (BP1-148, 5 mg/mL), and Lipid A or
MPL (5
mg/mL) were warmed to 55°C in a water bath for 15-20 minutes. BP1-148
lipopeptide has
the following structure: NHZ [STAPPAHGVTSAPDTRPAPGSTAPP(K-lipid
conjugated)G]-COOH. The following amounts of the warmed stock solutions were
added to
a clean stoppered 5 mL glass vial: 49.1 pL of DPPC, 103.5 ~L cholesterol, 60
pL of BP1-
148, 30 yL Lipid A and 657.4 pL of absolute ethanol. The mixture was vortexed
briefly (3
seconds x 7 times) and returned to the 55 °C water bath. One hundred
microliters of
deionized water (55 °C) was added into the vial and the was mixture
vortexed briefly as
above. The mixture was returned to the 55 °C water bath for 15-20
minutes, vortexing (as
above) twice during that period. Afterwards, the vials were cooled to room
temperature,
placed in a Dura-Stop MP shelf lyophilizer (FTS Systems, Stone Ridge, NY).
The foregoing results in a typical Lipomatrix at a lipid:solvent mass ratio of
1:47
after water was added at a solvent:water volume ratio of 9:1. After
lyophilization each vial
contained 15 mg of bulk lipid (at 50 Mol% cholesterol), 300 ~g of BP1-148 and
150 ~.g of
Lipid A. A typical lyophilization cycle, which was carried out under
microprocessor
control, is described below:
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Temp ( °C) Vacuum (mT) Duration (min)
-60 2000 240
-40 100 1440
-5 10 720
10 360
Example 2:
This example demonstrates that the inventive Lipomatrix does not produce
5 liposomes until the dried lipid preparation is hydrated. An aqueous phase,
at either a 9:1 or
7:I ethanol:water (v/v), was added to 0.297 mL of ethanol at 55°C
containing 14.8 mg
DPPC, 7.8 mg cholesterol, 0.2 mg MPL, and 0.11 mg MUC-1 peptide. A precipitate
formed
upon cooling to ambient temperature. The precipitate re-dissolved upon a two-
fold dilution
with ethanol/water at 9:1 or 7:1. This implies that the precipitate formed due
to lack of
solubility and was not necessarily liposomal.
Another 0.297 mL aliquot of the above mixture was mixed with an aqueous phase
containing carboxyfluorescein (CF) at a solvent:water volume ratio of 9:1 or
7:1. The total
fluorescence was measured and is shown in Figure 2. This represents what would
be expected
if liposomes were present entrapping 100% of the solute. This sample was
further diluted
i5 two-fold with saline, followed by five washes by centrifugation, resulting
in entrapment of 1
and 2% of the total CF at the 9:1 and 7:1 volume ratios, respectively. This is
lower than what
would be expected if liposomes were formed upon the initial mixing at 9:1 or
7:1, but what
would be expected if liposomes were formed during the dilution with excess
saline.
Freeze-fracture electron microscopy was performed on the liposomes made, as
described above, with ethanol mixed with saline at 9:1 or 7:1 (v/v). In
samples that were not
lyophilized, sheets of bilayers were observed, but no liposomal structures
were seen in the 9:1
(v:v) Lipomatrix formulation. See Figure 3, panel (a). Upon reconstitution of
a freeze-dried
preparation, however, liposomes were observed. See Figure 3, panel (b).
Similar results
were seen when the ethanol phase was mixed with the aqueous at a 7:1 volume
ratio.
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Example 3:
This example shows the effectiveness of various liposome preparations made
from
a Lipomatrix by the instant method in generating an immune response. The
Lipomatrix was
prepared, as outlined, by adding nine parts of an ethanol solution containing
lipid and
lipopeptide to one part water. The resultant liposomes contained, per 0.1 mL
dose, 10 pg
MUC-1 peptide (See, e.g., Koganty et al., DDT 1:190-98 (1996)), 20 pg MPL and:
MB-IX-1. 2 mg DMPC;
MB-IX-2. 2 mg DPPC;
MB-IX-3. 1.86 mg DMPC, 0.14 mg DMPG;
MB-iX-4. 1.86 mg DPPC, 0.14 mg DMPG;
MB-IX-5. 1.63 mg DPPC, 0.37 mg cholesterol; or
MB-IX-6. 1.6 mg DPPC, 0.125 mg DMPG, 0.375 mg
cholesterol.
Mice were immunized by subcutaneous injection in the inguinal area and
sacrificed nine days later. Lymph nodes and spleens were removed. Lymph node T
cells
were purified by passing through a nylon wool column. For the lymph node
assay, Antigen
Presenting Cells (APCs) were prepared by treatment of spleen cells from naive
mice with
Mitomycin C. For the cell proliferation assay, lymph node cells (with APCs)
and spleen cells
were incubated for four days with appropriate peptide antigens, followed by a
24 hour
incubation with Alamar Blue, after which the OD ratio at 610 to 570 nm was
measured. Prior
to the addition of Alamar Blue, supernatants of the cell proliferation assay
were harvested and
the gamma-interferon was measured in an Immuno-Fluorescence Assay (IFA). See
Ahmen et
al., J. Immunol. Methods 170:211-224.
Results are shown in figure 4. Single lipid formulations, such as samples MB-
IX-
1 and MB-IX-2 were not effective. In contrast, DPPC/cholesterol liposomal
preparations
induced a high IFN-y response, indicating a strong immune response. Control
experiments
confirm that these results are due neither to cholesterol itself nor to
liposomal size.
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Example 4:
This example demonstrates the importance of cholesterol to the immune response
induced by DPPC/cholesterol liposomes which were made according to the
invention.
Liposomes were prepared as in Example 3, with the following cholesterol
concentrations: 10,
20, 30, 40 and 50 Mol
As seen in Figure 5, DPPC/cholesterol formulations induce a strong immune
response with respect to lymphocyte IFN-gamma production. The response was
dependent on
the Mol % cholesterol in the formulation, with no biological response for
preparations containing
10 or 20 Mol % cholesterol.
Example 5:
This example shows the uniformity of hydrated preparations made using the
Lipomatrix process prepared with an ethanol:water volume ratio of 9:1.
Preparations were
analyzed using differential scanning calorimetry (DSC) and Raman vibrational
spectroscopy. The final product contained 13.1 mg/mL DPPC, 6.9 mg/mL
cholesterol
(i.e., 50 Mol%), 200 p,glmL Lipid A and 400 ~g/mL BP1-148.
DSC runs were performed on a Hart (now CSC) Scientific Model 7707 series
differential scanning microcalorimeter (Provo, UT) at 60 °C/hr. Fresh
aliquots were used
for each time point from a single sample vial hydrated at 55 °C. Each
run included a cell
containing normal saline solution for baseline determination. After baseline
subtraction and
correction for the thermal instrument response, calorimetric data were
analyzed to yield
excess heat content (pWatts) as a function of temperature, using software
supplied by Hart
Scientific. The calorimetric data were imported into Grams/32, v.5.0 (Galactic
Industries
Corporation, Salem, NH) for baseline and offset correction, smoothing and
plotting. Data
were not smoothed or only minimally smoothed by using a Savitsky-Golay
smoothing
routine. This method uses a convolution approach and performs a least squares
fit to a
specified window. The data was smoothed using a 3rd order polynomial and a
window of
5-11 data points.
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Raman vibrational spectroscopy was collected at room temperature using Raman
microspectroscopy. For lyophilized powders, an argon laser was focused to a 1-
2 ~m spot
(S 14 nm excitation, xS0 objective). For hydrated preparations, samples were
packed in a
glass capillary by centrifugation for 1S minutes at room temperature in a
hematocrit
centrifuge. Raman spectra were again collected using a xS0 objective, but the
laser was
defocused 80 % to prevent local heating of the bilayer structures. In both
cases, power at
the laser head was set to 300 mW and reduced to 2S % at the microscope. The
Raman
signal was dispersed by the spectrometer (1800gr/mm grating) onto a CCD
detector.
Typically 10 spectra were coadded using a time constant of 30 - 60 seconds per
collection.
Spectral resolution was at " 1 cm'' .
As shown in Figure 6, the DSC profile for a liposomal preparation made by the
Lipomatrix process as outlined in Example 1 revealed a very flat endotherm
that did not
change in time. For comparison, the DSC heating profile of liposomes comprised
of DPPC
alone exhibits two prominent transitions at 38 °C (pretransition) and
41 °C (main
1S transition). The absence of these transitions in the Lipomatrix
formulations at SO Mol%
cholesterol indicates that the components are devoid of DPPC-rich domains and
is indicative
of a liposomal preparation in which the components are well mixed. To further
characterize
the uniformity of the Lipomatrix formulations at a molecular level, Raman
vibrational
spectroscopy was used. Figure 7A shows that at two different sites (solid and
dotted lines)
in the lyophilized film there was no significant difference in the relative
concentrations of
cholesterol to DPPC. Moreover, the same relative ratios of cholesterol to DPPC
was also
observed in the hydrated product (Figure 7B).
Example 6:
2S
This example demonstrates that the present method retains utility on a larger
scale.
A 120 mL batch was prepared by the Lipomatrix process of Liposornal MUC-1
vaccine at
1S mg bulk lipid (at SO Mol% cholesterol), 300 pg BP1-148, 1S0 ~,g Lipid A per
vial as
outlined in example 1 and filtered at room temperature one hour after
production {MB-
XLIV-B) and eight hours after production (MB-XLIV-A). As shown in the tables
below, no
detectable losses were observed by HPLC.
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HPLC Results of 120 mL Scale-up Formulation (MB-XLIV-A and MB-XLIV-B)
DPPC Cholesterol BPl-148 Lipid
A
Sample' (mg) (mg) (!gig) (wg)
Expected Values 9.825 5.175 300 150
MB-XLIV-A, initial 9.82 4.17 298 187
MB-XLIV-A, t=0 hr filtered9.85 4.12 297 196 .
MB-XLIV-A, t = 8 hr filtered10.35 4.49 314 172
MB-XLIV-B, initial 9.29 4.17 300 188
MB-XLIV-B, t=0 hr filtered9.52 4.27 290 178
'Values represent the average of duplicates
Example 7:
This Example illustrates the use of the present Lipomatrix formulations in
preparing
a tumor antigen-specific cancer vaccine. A Liposomal MUC-1 vaccine was
prepared, as in
Example 1, which contained 15 mg bulk lipid (at 50 Mol% cholesterol), 300 pg
BP1-148,
150 pg Lipid A per vial. The following parameters were varied:
a. the alcohol, ethanol or tent-butanol;
b. the solvent to water ratio; and
c. the lipid to solvent mass ratio.
Briefly, samples were hydrated at 55 °C, cooled to room temperature and
injected
into mice as in Example 4. The table below shows that strong IFN-y responses
were
observed under almost all variations, particularly at the higher solvent:lipid
mass ratios.
The benefit of a higher solvent:lipid mass ratio is at least two-fold. First,
by increasing the
amount of solvent the fill volume could be increased (making production
easier). Second,
the higher solvent: lipid mass ratios allowed room temperature filtering and
filling, a great
asset for scaleability (vide infra). The table also indicates that filtration
following eight
hours (as in Example 6) does not adversely effect activity.
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S:W L:S IFN-'y
(ng/mL)
Solvent (vol.) (mass) LN SPL Total
E 9:1 1:30 9.9 5.3 15.2
E 7:1 1:30 0.6 4.5 5.1
E' 9:1 1:47 3.0 13.1 16.1
E 9:1 1:47 2.2 19.0 21.2
B 9:1 1:17 0.3 0 0.3
B 7:1 1:17 3.1 5.2 8.3
B 9:1 1:30 2.4 14.9 17.3
B ~ 7:1 1:3 3.9 4.6 8.5
0
E= ethanol; B= tert-butanol; L= lipid; S= solvent; W= water; LN = lymph node;
SPL
= spleen; *filtration at t=8 hr after mixing.
Example 8:
This example exhibits the stability of the lyophilized Lipomatrix. A liposomal
MUC-1 vaccine was prepared, which had 15 mg bulk lipid (at 50 Mol %
cholesterol), 300
pg BP1-148, 150 pg Lipid A per vial by the method of Example 1, and analyzed
at time
zero and after 3 and 6 months to determine the stability by HPLC analysis. The
Designations A and B are as defined in Example 6. As shown below, no
significant
changes were observed from the initial time point:
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Physical Stability Studies of 9:1 Lipomatrix Formulation
Method t = 6 monthst = 3 monthst = 0 Ex ected Value
PSS770 A 3.71 pm 3.64 pm 4.00 pm
Sizin
g
B 3.44 pm 3.64 pm 3.56 pm
pH A 4.2 4.10 4.19
B 4.2 4.19 4.24
Appearance A Thin white Thin white Thin white
film film film
B Thin white Thin white Thin white
film film film
HPLC:
A 10.1 m 9.54 m 9.80 m 9.825 m
DPPC B 9.7 m 9.01 m 8.99 m 9.825 m
A 5.2 m 4.89 m 5.14 m 5.175 m
Chol B 5.3 m 4.82 m 5.10 m 5.175 m
A 123 wg 130 ~g 122 p.g 150 p,g
Lipid A B 132 ~g 150 ~g 150 pg 150 pg
A 275 ~.g 282 Egg 278 pg 300 pg
BPl-148 B 278 ~.g 278 ~g 274 pg 300 wg
Example 9:
This Example that the present hydrated Lipomatrix formulations are stable at
room
temperature. The Lipomatrix process was used to make several vials of
Liposomal MUC-1
vaccine with 15 mg bulk lipid (at 50 Mol% cholesterol), 150 p,g BP1-148, 150
~,g Lipid A
per vial, using an ethanol solvent:water ratio of 9:1 by volume.
Mice were subcutaneously injected with these preparations after room
temperature
storage of the hydrated preparations for 0, 2, 4 and 24 hours. Two sets of
four mice were
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injected at each time point. The averages shown in Figure 8 demonstrate a
stable product
following hydration at all time points.
*******
The foregoing detailed description and examples are presented merely for
illustrative
purposes and are not meant to be limiting. Thus, one skilled in the art will
readily recognize
additional embodiments within the scope of the invention that are not
specifically exemplified.
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