Note: Descriptions are shown in the official language in which they were submitted.
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SUGAR GLASSIFIED VIRUS LIKE PARTICLES (VLPS)
FIELD OF THE INVENTION
[001] This invention relates to stabilizing virus like particles (VLPs) by
associating said VLPs
with a sugar glass.
BACKGROUND
[002] The majority of commonly recommended vaccines require storage
temperatures of 35 F-
46 F (2 C-8 C) and must not be exposed to freezing temperatures nor
temperatures greater than
room temperature (about 22 C - 25 C). Introduction of varicella vaccine in
1995 and of the live
attenuated influenza vaccine (LAIV) more recently increased the complexity of
vaccine storage.
Both varicella vaccine and LAIV must be stored in a continuously frozen state
<5 F (-15 C) with
no freeze-thaw cycles.
[003] In recent years, instances of improper vaccine storage have been
reported. An estimated
17%-37% of providers expose vaccines to improper storage temperatures and
refrigerator
temperatures are more commonly kept too cold than too warm. Freezing
temperatures can
irreversibly reduce the potency of vaccines required to be stored at 35 F-46 F
(2 C-8 C).
Certain freeze-sensitive vaccines contain an aluminum adjuvant that
precipitates when exposed
to freezing temperatures. This may result in loss of the adjuvant effect and
vaccine potency.
Physical changes are not always apparent after exposure to freezing
temperatures and visible
signs of freezing are not necessary to result in a decrease in vaccine
potency. Although the
potency of the majority of vaccines can be affected adversely by storage
temperatures that are
too warm, these effects are usually more gradual, predictable, and smaller in
magnitude than
losses from temperatures that are too cold. In contrast, varicella vaccine and
LAW are required
to be stored in continuously frozen states and lose potency when stored above
the recommended
temperature range.
[004] Virus like particles (VLPs) are useful as vaccines against diseases
(e.g. influenza). Virus-
like particles (VLPs) closely resemble mature virions, but they do not contain
viral genomic
material (i.e., viral genomic RNA). Therefore, VLPs are nonreplicative in
nature, which make
them safe for administration in the form of an immunogenic composition (e.g.,
vaccine). In
addition, VLPs can express envelope glycoproteins on the surface of the VLP,
which is the most
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physiological configuration. Moreover, since VLPs resemble intact virions and
are multivalent
particulate structures, VLPs may be more effective in inducing neutralizing
antibodies to the
envelope glycoprotein than soluble envelope antigens. Further, VLPs can be
administered
repeatedly to vaccinated hosts, unlike many recombinant vaccine approaches.
However, like the
varicella vaccine and LAIV, VLPs are sensitive to temperature changes and this
must be
maintained in a controlled environment. The ideal VLP formulation would resist
high (greater
that about 25 C) and low temperatures (less than about 2 C), to facilitate
distribution.
[005] Current methods for avoiding temperature associated degradation of
vaccines are
inadequate. For example, live-attenuated vaccines (and some non-live vaccines)
are often
lyophilized because of their intrinsic instability. The lyophilized products
are reconstituted with
diluent immediately before administration. Because lyophilization is a time-
consuming and
capacity-limiting step of vaccine production, lyophilized vaccines are usually
presented in multi-
dose vials. Some global guidelines require that unused vaccines in a multi-
dose vial be discarded
within six hours of reconstitution due to the concerns of potential
contamination and potency
loss. This results in vaccine wastage, which can account for losses of 50% or
more of the
vaccine doses distributed.
[006] In the public health arena, a common approach for freeze-prevention is
to strengthen and
optimize the cold chain. Drawbacks of this approach include the expense
associated with
extending, updating and improving the cold chain equipment, monitoring the
equipment, and
training those using the equipment. Moreover, although cold chain improvement
can minimize
freeze-damage, such improvement will not eliminate the occurrence of freeze-
damage.
Additionally, cold chain improvements will not mitigate the freezing that
occurs outside of the
cold chain in colder climates.
[007] There remains a need for compositions and methods for stabilizing
temperature-sensitive
vaccines, and specifically for compositions and methods for stabilizing
vaccines.
SUMMARY OF THE INVENTION
[008] The present invention comprises a composition comprising at least one
virus like particle
(VLP) and a sugar glass. In one embodiment, said sugar glass is composed of a
monosaccharide
and/or disaccharide. In another embodiment, said monosaccharide is selected
from the group
consisting of glucose, sorbitol, galactose, mannose, and mannitol. In another
embodiment, said
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disaccharide is selected from the group consisting of trehalose, maltose,
maltotriose, lactose,
lactulose, and sucrose. In another embodiment, said VLP and said sugar glass
is a powder. In
another embodiment, said powder is made by spray drying, freeze drying,
milling or a
combination thereof. In another embodiment, said powder has a mean particle
diameter between
about 0.1 nm to about 100 microns. In another embodiment, said powder is
suspended in a non-
aqueous solvent that will not dissolve said sugar glass. In another
embodiment, said non-
aqueous solution is selected from the group consisting of triacetin, isopropyl
myristate, medium
chain triglycerides, short, medium, long-chain mono-, di-, tri-, glycerides,
aliphatic and aromatic
alcohols or a combination thereof. In another embodiment, said powder is
administered to an
animal orally, via inhalation, intradermally, intranasally, intramusclarly,
intraperitoneally,
intravenously, subcutaneously, or mucosally (e.g. sublingually or buccally).
In another
embodiment, said VLP comprises at least one viral protein. In another
embodiment, said vial
protein is from influenza, RSV and/or VZV. In another embodiment, said VLP has
increased
stability when compared to VLP that is not associated with a sugar glass.
[009] The present invention also comprises, a composition comprising at least
one VLP and a
sugar glass, wherein said VLP has increased stability when compared said
composition without
sugar glass. In another embodiment, said increased stability is increase
thermal stability. In
another embodiment, said sugar glass is composed of a monosaccharide or
disaccharide. In
another embodiment, wherein said monosaccharide is selected from the group
consisting of
glucose, sorbitol, galactose, mannose, and mannitol. In another embodiment,
said disaccharide is
selected from the group consisting of trehalose, maltose, maltotriose,
lactose, lactulose, and
sucrose. In another embodiment, said stabilized sugar glass VLP is a powder.
[010] The present invention also comprises, a dry powder formulation
comprising a VLP and a
sugar glass. In one embodiment, said sugar glass is composed of a
monosaccharide or
disaccharide. In another embodiment, said monosaccharide is selected from the
group
consisting of glucose, sorbitol, galactose, mannose, and mannitol. In another
embodiment, said
disaccharide is selected from the group consisting of trehalose, maltose,
maltotriose, lactose,
lactulose, and sucrose. In another embodiment, said dry powder is made by
spray drying,
milling or a combination thereof. In another embodiment, said powder is
administered to an
animal orally, via inhalation, intradermally, intranasally, intramusclarly,
intraperitoneally,
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intravenously, or subcutaneously. In another embodiment, said powder is
administered to an
animal via inhalation using an inhaler and/or subcutaneously using a jet of
high-pressure air.
[011] The present invention also comprises, a method of delivering a sugar
glass stabilized
VLP comprising reconstituting a solid form of sugar glassified VLPs in a
solvent and
administering said reconstituted VLPs into an animal. In one embodiment, said
powder is
administered to an animal orally, via inhalation, intradermally, intranasally,
intramusclarly,
intraperitoneally, intravenously, or subcutaneously.
[012] The present invention also comprises, a method of delivering a sugar
glass stabilized
VLP comprising administering a solid form of sugar glassified VLPs via
inhalation and/or
injection.
BRIEF DESCRIPTION OF THE FIGURES
[013] Figure IA SDS PAGE and western blot of VLPS before the glassification of
VLPs
[014] Figure 113 SDS PAGE and western blot of VLPS after the glassification of
VLPs
[015] Figure 2 SDS PAGE and western blot of VLPS before and after the
glassification of
VLPs
DETAILED DESCRIPTION
[016] As used herein, the term "adjuvant" refers to a compound that, when used
in combination
with a specific immunogen (e.g. a VLP) in a formulation, will augment or
otherwise alter or
modify the resultant immune response. Modification of the immune response
includes
intensification or broadening the specificity of either or both antibody and
cellular immune
responses. Modification of the immune response can also mean decreasing or
suppressing
certain antigen-specific immune responses.
[017] As used herein, the term "ambient" temperatures or conditions are those
at any given
time in a given environment. Typically, ambient room temperature is
approximately 22 C,
ambient atmospheric pressure, and ambient humidity are readily measured and
will vary
depending on the time of year, weather conditions, altitude, etc.
[018] As used herein, the term "buffer" refers to a buffered solution that
resists changes in pH
by the action of its acid-base conjugate components. The pH of the buffer will
generally be
chosen to stabilize the active material of choice, and will be ascertainable
by those in the art.
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Generally, this will be in the range of physiological pH, although some
proteins, can be stable at
a wider range of pHs, for example acidic pH. Thus, preferred pH ranges are
from about 1 to
about 10, with from about 3 to about 8, from about 6.0 to about 8.0, from
about 7.0 to about 7.4,
and from about 7.0 to about 7.2. Suitable buffers include a pH 7.2 phosphate
buffer and a pH 7.0
citrate buffer. As will be appreciated by those in the art, there are a large
number of suitable
buffers that may be used. Suitable buffers include, but are not limited to,
amino acids, potassium
phosphate, sodium phosphate, sodium acetate, histidine-HC1, sodium citrate,
sodium succinate,
ammonium bicarbonate and carbonate. Generally, buffers are used at molarities
from about 1
mM to about 2 M, with from about 2 mM to about 1 M being preferred, and from
about 10 mM
to about 0.5 M being especially preferred, and 25 to 50 mM being particularly
preferred.
[019] As used herein, the term "effective dose" generally refers to that
amount of VLPs
sufficient to induce immunity, to prevent and/or ameliorate an infection or to
reduce at least one
symptom of an infection and/or to enhance the efficacy of another dose of a
VLP. An effective
dose may refer to the amount of VLPs sufficient to delay or minimize the onset
of an infection.
An effective dose may also refer to the amount of VLPs that provides a
therapeutic benefit in the
treatment or management of an infection. Further, an effective dose is the
amount with respect
to VLPs of the invention alone, or in combination with other therapies, that
provides a
therapeutic benefit in the treatment or management of an infection. An
effective dose may also
be the amount sufficient to enhance a subject's (e.g., a human's) own immune
response against a
subsequent exposure to an infectious agent. Levels of immunity can be
monitored, e.g., by
measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by
plaque
neutralization, complement fixation, enzyme-linked immunosorbent, or
microneutralization
assay. In the case of a vaccine, an "effective dose" is one that prevents
disease and/or reduces
the severity of symptoms.
[020] As used herein, the term "excipients" generally refer to compounds or
materials that are
added to increase the stability of a therapeutic agent during glassification
(by, e.g., the spray
freeze dry process) and afterwards, for long-term storage. Suitable excipients
can be, e.g., agents
that do not thicken or polymerize upon contact with water, are basically
innocuous when
administered to a patient and does not significantly interact with the
therapeutic agent in a
manner that alters its biological activity. Suitable excipients are described
below and include,
but are not limited to, proteins such as human and bovine serum albumin,
gelatin, carbohydrates,
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sugar alcohols, e.g. glycerin, erythritol, glycerol, arabitol, xylitol,
sorbitol, and mannitol,
propylene glycol, Pluronics, surfactants and combinations thereof. Excipients
can be
multifunctional constituents of solutions or suspensions of invention.
[021] As used herein, the term "effective amount" refers to an amount of VLPs
necessary or
sufficient to realize a desired biologic effect. An effective amount of the
composition would be
the amount that achieves a selected result, and such an amount could be
determined as a matter
of routine by a person skilled in the art. For example, an effective amount
for preventing,
treating and/or ameliorating an infection could be that amount necessary to
cause activation of
the immune system, resulting in the development of an antigen specific immune
response upon
exposure to VLPs of the invention. The term is also synonymous with
"sufficient amount."
[022] As use herein, the term "infectious agent" refers to microorganisms that
cause an
infection in a vertebrate. Usually, the organisms are viruses, bacteria,
parasites and/or fungi.
[023] As used herein, the term "glass" or "glassy state" or "glassy matrix,"
refers to a liquid
that has a markedly reduced ability to flow, i.e. it is a liquid with a very
high viscosity, wherein
the viscosity ranges from 1010 to 1014 pascal-seconds. It can be viewed as a
metastable
amorphous system in which the molecules have vibrational motion but have very
slow (almost
immeasurable) rotational and translational components. As a metastable system,
it is stable for
long periods of time when stored well below the glass transition temperature.
Because glasses
are not in a state of thermodynamic equilibrium, glasses stored at
temperatures at or near the
glass transition temperature relax to equilibrium and lose their high
viscosity. The resultant
rubbery or syrupy, flowing liquid is often chemically and structurally
destabilized. While a glass
can be obtained by many different routes, it appears to be physically and
structurally the same
material by whatever route it was taken. The process used to obtain a glassy
matrix for the
purposes of this invention is generally a solvent sublimation and/or
evaporation technique.
[024] As used herein, the term "glass transition temperature" is represented
by the symbol Tg
and is the temperature at which a composition changes from a glassy or
vitreous state to a syrup
or rubbery state. Generally, Tg is determined using differential scanning
calorimetry (DSC) and
is standardly taken as the temperature at which onset of the change of heat
capacity (Cp) of the
composition occurs upon scanning through the transition. The definition of Tg
is always
arbitrary and there is no present international convention. The Tg can be
defined as the onset,
midpoint or endpoint of the transition; for purposes of this invention we will
use the onset of the
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changes in Cp when using DSC and DER. See the article entitled "Formation of
Glasses from
Liquids and Biopolymers" by C. A. Angell: Science, 267, 1924-1935 (Mar. 31,
1995) and the
article entitled "Differential Scanning Calorimetry Analysis of Glass
Transitions" by Jan P.
Wolanczyk: Cryo-Letters, 10, 73-76 (1989). For detailed mathematical treatment
see "Nature of
the Glass Transition and the Glassy State" by Gibbs and DiMarzio: Journal of
Chemical Physics,
28, NO. 3, 373-383 (March, 1958). These articles are incorporated herein by
reference.
[025] As used herein, the term "stable" formulation or composition is one in
which the
biologically active material therein (e.g. VLPs) essentially retains its
physical stability and/or
chemical stability and/or biological activity upon storage. Various analytical
techniques for
measuring stability are available in the art and are reviewed, e.g., in
Peptide and Protein Drug
Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs.
(1991) and
Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured
at a selected
temperature for a selected time period. Trend analysis can be used to estimate
an expected shelf
life before a material has actually been in storage for that time period.
[026] As used herein, the term "vaccine" refers to a formulation which
contains VLPs which is
in a form that is capable of being administered to a vertebrate and which
induces a protective
immune response sufficient to induce immunity to prevent and/or ameliorate an
infection and/or
to reduce at least one symptom of an infection and/or to enhance the efficacy
of another dose of
VLPs. Upon introduction into a host, the vaccine is able to provoke an immune
response
including, but not limited to, the production of antibodies and/or cytokines
and/or the activation
of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic
cells and/or other cellular
responses. In one embodiment, VLPs vaccines are associated with sugar glass
and/or were
stabilized with sugar glass.
[027] As used herein, the term "virus-like particle" (VLP) refers to a
structure that in at least
one attribute resembles a virus but which has not been demonstrated to be
infectious. Virus-like
particles in accordance with the invention do not carry genetic information
encoding for the
proteins of the virus-like particles. In general, virus-like particles lack a
viral genome and,
therefore, are noninfectious. In addition, virus-like particles can often be
produced in large
quantities by heterologous expression and can be easily purified.
[028] Vaccines or drugs in solution ready for injections are inherently
unstable. Methods to
tackle this instability are known in the art (e.g. freeze drying). However,
this is an inconvenient
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and inherently dangerous, since incorrect reconstitution or dried vaccines or
drugs can result in
wrong doses or contaminated solution and freezing and/or thawing of a vaccine
or drug can also
result in wrong dosing. Thus, there is a need for new improved vaccine
formulations that makes
vaccine delivery easier and safer, decrease dependency on the cold chain
and/or increase thermal
stability of vaccines and/or reduce the number of immunizations interventions.
Such vaccine
formulations would make distribution of vaccines worldwide more efficient and
economical.
[029] One of the keystones in the improvement of vaccine formulations is
obtaining stable
antigens in the dry state. The most commonly used method for preparing solid
proteinaceous
drug is freeze-drying (lyophilization). However, during freeze-drying the
proteinaceous drug is
subjected to freezing and drying stress by which activity can be lost.
Therefore, a protective
agent is required to prevent damaging effects of lyophilization.
[030] It is known that carbohydrates can protect various types of drug
substances like proteins
and vaccines during freezing, drying and storage. If dried properly, a
proteinaceous drug can be
incorporated in a matrix consisting of carbohydrate in the amorphous glassy
state (sugar glass).
The stabilizing effect of these sugar glasses has been explained by the
formation of a matrix that
strongly reduces diffusion and molecular mobility and acts as a physical
barrier between
particles or molecules. Both the lack of mobility and physical barrier
provided by the glass
matrix, prevent aggregation and degradation of the dried material (Amorij, JP
et at. (2007)
Vaccine, 25, 6447-6457).
[031] To make sugar glass, the sugar must be dried below the glass transition
temperature of a
carbohydrate, e.g. a sugar. The glass transition temperature is the
temperature below which the
physical properties of amorphous materials vary in a manner similar to those
of a solid phase
(glassy state), and above which amorphous materials behave like liquids
(rubbery state). A
material's glass transition temperature, Tg, is the temperature below which
molecules have little
relative mobility. Tg is usually applicable to wholly or partially amorphous
phases such as
glasses and plastics. Above Tg, the secondary, non-covalent bonds between the
polymer chains
become weak in comparison to thermal motion, and the polymer becomes rubbery
and capable
of elastic or deformation without fracture.
[032] Sugar glass is made when drying a sugar mixture below the Tg
temperature. As the sugar
solution containing an active molecule is dried, it can either crystallize
when the solubility limit
of the sugar is reached or can become a supersaturated syrup. The ability of
the sugar to resist
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crystallization is a crucial property of a good stabilizer. Trehalose is good
is known to make
glass (Green J L. & Angel C A. Phase relations and vitrification in saccharide
water solutions
and the trehalose anomaly J. Phys. Chem. 93 2880-2882 (1989)) but is not
unique. Further
drying progressively solidifies the syrup, which turns into a glass at low
residual water content.
Imperceptibly, the active molecules change from liquid solution in the water
to solid solution in
the dry sugar glass. Chemical diffusion is negligible in a glass and therefore
chemical reactions
virtually cease. Since denaturation is a chemical change it cannot occur in
the glass and the
molecules are stabilized.
[033] Thus, the invention comprises a composition comprising at least one
virus like particle
(VLP) associated and a sugar glass. In one embodiment, said VLP is encased by
said sugar
glass. In one embodiment, said VLP associates with said sugar glass. In one
embodiment, said
sugar glass is composed of a monosaccharide and/or disaccharide. In another
embodiment, said
monosaccharide is selected from the group consisting of glucose, sorbitol,
galactose, mannose,
and mannitol. In another embodiment, said disaccharide is selected from the
group consisting of
trehalose, maltose, maltotriose, lactose, lactulose, and sucrose. Other sugars
include galactose,
mannose, xylose, sorbose, lactose, palactose, raffinose, maltodextrins,
melezitose, maltose,
fructose, arabinose, xylose, ribose, rhamnose, xylitol, erythritol, threitol,
gluconate, and/or the
like. The suspension or solution can also include, e.g., a polymer, such as
starch, starch
derivatives, carboxymethyl starch, hydroxyethyl starch (HES), and/or dextran.
[034] In another embodiment of the invention, VLPs are stable in ambient
temperatures, when
said VLPs are associated with a sugar glass. Said sugar is preferably one
which does not
crystallize at freezing temperatures such that it destabilizes said VLPs in a
glassy formulation.
The amount of sugar used in the suspension or solution can vary depending on
the nature of the
VLP, the type of sugar, and the intended use. However, generally, the final
concentration of the
sugar is between about 1% and 40%; more preferably, between about 1% and 20%
by weight. In
one embodiment, the suspension or solution comprises about 60 mg/ml Trehalose.
In another
embodiment, the suspension or solution comprises about 50 mg/ml of Mannitol.
In another
embodiment, the suspension or solution comprises about 30 mg/ml of Trehalose
and about 25
mg/ml of Mannitol. In another embodiment, the suspension or solution comprises
about 30
mg/ml of Trehalose and about 30 mg/ml of Mannitol.
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[035] In one embodiment, said VLP and sugar glass composition may be a powder.
In another
embodiment, said powder is made by spray drying, freeze-drying, milling or a
combination
thereof. In other embodiment, said powder has a mean particle diameter between
about 0.1 nm
to about 100 microns. Spray drying and freeze drying is well known in the art
(see 6,372,258,
7,258,873, and 5,230,162, all of which are herein incorporated by reference in
their entireties.)
[036] The formulation of the invention comprise virus-like particles (VLPs)
and a sugar glass.
Said VLPs comprise at least a viral core protein (e.g. Influenza M1,
retrovirus gag, RSV M,
Newcastle disease M etc.) and at least one viral surface envelope protein
(e.g. influenza HA
and/or NA, HIV gp120, RSV F, Newcastle disease F). Chimeric VLPs are VLPs
having at least
two proteins in the VLP, wherein one protein can drive VLP formation (e.g.
matrix protein) and
the other protein is from a heterologous infectious agent. In one embodiment,
said infectious
agent proteins may have antigenic variations of the same protein. In another
embodiment, said
infectious agent protein is from an unrelated agent. In another embodiment,
said chimeric VLPs
comprise a chimeric protein (fusion protein) comprising the antigenic portion
of one protein
fused to the transmembrane and/or cytoplasmic region of a different
(heterologous) protein (See
U.S. applications 60/902,337, filed February 21, 2007, and 60/970,592, filed
September 7, 2007,
both of which are incorporated herein by reference in their entireties for all
purposes.
[037] Infectious agents can be viruses, bacteria and/or parasites. A protein
that may be
expressed on the surface of VLPs can be derived from viruses, bacteria and/or
parasites. The
proteins derived from viruses, bacteria and/or parasites can induce an immune
response (cellular
and/or humoral) in a vertebrate that which will prevent, treat, manage and/or
ameliorate an
infectious disease in said vertebrate.
[038] Non-limiting examples of viruses from which said infectious agent
proteins can be
derived from are the following: influenza (A and B, e.g. HA and/or NA),
coronavirus (e.g.
SARS), hepatitis viruses A, B, C, D & E3, human immunodeficiency virus (HIV),
herpes viruses
1, 2, 6 & 7, cytomegalovirus, varicella zoster, papilloma virus, Epstein Barr
virus, parainfluenza
viruses, adenoviruses, bunya viruses (e.g. hanta virus), coxsakie viruses,
picoma viruses,
rotaviruses, rhinoviruses, rubella virus, mumps virus, measles virus, Rubella
virus, polio virus
(multiple types), adeno virus (multiple types), parainfluenza virus (multiple
types), avian
influenza (various types), shipping fever virus, Western and Eastern equine
encephalomyelitis,
Japanese encephalomyelitis, fowl pox, rabies virus, slow brain viruses, rous
sarcoma virus,
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Papovaviridae, Parvoviridae, Picomaviridae, Poxviridae (such as Smallpox or
Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus),
Togaviridae (e.g.,
Rubivirus), Newcastle disease virus, West Nile fever virus, Tick borne
encephalitis, yellow
fever, chikungunya virus, and dengue virus (all serotypes).
[039] In another embodiment, the specific proteins from viruses may comprise:
HA and/or NA
from influenza virus (including avian), S protein from coronavirus, gp160,
gp140 and/or gp4l
from HIV, gp Ito IV and Vp from varicella zoster, E and preM/M from yellow
fever virus,
Dengue (all serotypes) or any flavivirus. Also included are any protein from a
virus that can
induce an immune response (cellular and/or humoral) in a vertebrate that can
prevent, treat,
manage and/or ameliorate an infectious disease in said vertebrate. In one
embodiment, said VLP
comprises at least one influenza protein. In another embodiment, said
influenza protein is HA or
NA. In another embodiment, said VLP comprises at least one RSV protein. In
another
embodiment, said RSV protein is RSV M, F and/or G. In another embodiment, said
VLP
comprises a VZV protein. In another embodiment, said VZV protein is gE and/or
at least one
tegument protein.
[040] Non-limiting examples of bacteria from which said infectious agent
proteins can be
derived from are the following: B. pertussis, Leptospira pomona, S. paratyphi
A and B, C.
diphtheriae, C. tetani, C. botulinum, C. perfringens, C. feseri and other gas
gangrene bacteria, B.
anthracis, P. pestis, P. multocida, Neisseria meningitidis, N. gonorrheae,
Hemophilus
influenzae, Actinomyces (e.g., Norcardia), Acinetobacter, Bacillaceae (e.g.,
Bacillus anthrasis),
Bacteroides (e.g., Bacteroidesfragilis), Blastomycosis, Bordetella, Borrelia
(e.g., Borrelia
burgdorferi), Brucella, Campylobacter, Chlamydia, Coccidioides,
Corynebacterium (e.g.,
Corynebacterium diptheriae), E. coli (e.g., Enterotoxigenic E. coli and
Enterohemorrhagic E.
coli), Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae
(Klebsiella, Salmonella
(e.g., Salmonella typhi, Salmonella enteritidis, Serratia, Yersinia,
Shigella), Erysipelothrix,
Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella
(e.g., Legionella
pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma,
Mycobacterium
(e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g.,
Vibrio cholerae),
Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa),
Rickettsiaceae,
Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella
spp.,
Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae
and Groups
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A, B, and C Streptococci), Ureaplasmas. Treponemapollidum, Staphylococcus
aureus,
Pasteurella haemolytica, Corynebacterium diptheriae toxoid, Meningococcal
polysaccharide,
Bordetella pertusis, Streptococcus pneumoniae, Clostridium tetani toxoid, and
Mycobacterium
bovis.
[041] Non-limiting examples of parasites from which said infectious agent
proteins can be
derived which are the causative agent for following: leishmaniasis (Leishmania
tropica
mexicana, Leishmania tropica, Leishmania major, Leishmania aethiopica,
Leishmania
braziliensis, Leishmania donovani, Leishmania infantum, Leishmania chagasi),
trypanosomiasis
(Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense), toxoplasmosis
(Toxoplasma gondii) , schistosomiasis (Schistosoma haematobium,
Schistosomajaponicum,
Schistosoma mansoni, Schistosoma mekongi, Schistosoma intercalatum), malaria
(Plasmodium
virax, Plasmodiumfalciparium, Plasmodium malariae and Plasmodium ovale)
Amebiasis
(Entamoeba histolytica), Babesiosis (Babesiosis microti), Cryptosporidiosis
(Cryptosporidium
parvum), Dientamoebiasis (Dientamoeba fragilis), Giardiasis (Giardia lamblia),
Helminthiasis
and Trichomonas (Trichomonas vaginalis). The above lists are meant to be
illustrative and by no
means are meant to limit the invention to those particular bacterial, viral or
parasitic organisms.
[042] Another embodiment of the invention comprises a composition comprising
at least one
VLP in a sugar glass, wherein said VLP has increased stability when compared
to VLP that is
not in a sugar glass. In one embodiment, said increased stability is increase
thermal stability and
increased stability in ambient temperature. VLPs are "stable" in a
pharmaceutical composition
if, e.g., said VLP shows no significant increase in aggregation, precipitation
and/or denaturation
upon visual examination of color and/or clarity, as measured by UV light
scattering or by size
exclusion chromatography, or any biological assay. A "stable" formulation or
composition is
one in which the biologically active material therein essentially retains its
physical stability
and/or chemical stability and/or biological activity upon storage. Various
analytical techniques
for measuring stability are described below and available in the art and are
reviewed, e.g., in
Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker,
Inc., New York,
N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).
Stability can be
measured at a selected temperature for a selected time period. Trend analysis
can be used to
estimate an expected shelf life before a material has actually been in storage
for that time period.
For example, the composition can be stable at room temperature (about 25 C)
for at least 3
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months, and/or stable at about 2-8 C for at least 1 year. Furthermore, the
composition can be
stable following freezing (to, e.g., -70 C) and thawing of the composition. In
another
embodiment, said sugar glass is composed of a monosaccharide or disaccharide.
In another
embodiment, said monosaccharide is selected from the group consisting of
glucose, sorbitol,
galactose, mannose, and mannitol. In another embodiment, said disaccharide is
selected from
the group consisting of trehalose, maltose, maltotriose, lactose, lactulose,
and sucrose. In
another embodiment, said stabilized sugar glass VLP is a powder.
[043] The VLP associated sugar glass composition may have additional
exceptions that help
stabilize said VLPs. Thus, the present invention includes compositions, such
as suspensions or
solutions of VLPs, a polymer additive, an amino acid additive, and/or a
surfactant, to help
improved stability. The suspensions or solutions can include other ingredients
(i.e. excipients)
such as buffers, carriers, preservatives, colloidal stabilizers, fillers,
diluents, lubricants,
amphiphiles, and/or stabilizers. For example, polymers can be included in the
suspensions or
solutions of the method, e.g., to provide protective and structural benefits.
The linear or
branching strands of polymers can provide, e.g., increased structural strength
to the particle
compositions of the invention. Polymers can be applied as a protective and/or
time release coat
to the outside or powder particles of the invention. Many polymers such as
polyvinyl
pyrrolidone, polyethylene glycol, poly amino acids, such poly L-lysines, can
significantly
enhance reconstitution rates in aqueous solutions. Polymer protective agents,
in the methods of
the invention can include, e.g., starch and starch derivatives, such as
oxidized starch,
carboxymethyl starch and hydroxyethyl starch (HES). Others include hydrolyzed
gelatin,
unhydrolyzed gelatin, ovalbumin, collagen, chondroitin sulfate, a sialated
polysaccharide, actin,
myosin, microtubules, dynein, kinetin, human serum albumin, and/or the like.
Other excipients
can be included in the formulation. For example, amino acids, such as arginine
and methionine
can be constituents of the formulation and compositions. The amino acids can,
e.g., act as
zwitterions that block charged groups on processing surfaces and storage
containers preventing
nonspecific binding of bioactive materials. The amino acids can increase the
stability of
compositions by, e.g., scavenging oxidation agents, scavenging deamidation
agents, and
stabilizing the conformations of proteins. In another example, glycerol can be
included in the
formulations of the invention, e.g., to act as a plasticizer in the powder
particle compositions.
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EDTA can be included in the composition, e.g., to reduce aggregation of
formulation
constituents and/or to scavenge metal ions that can initiate destructive free
radical chemistries.
[044] VLP and sugar glass composition of the invention may also include, e.g.,
a surfactant
compatible with the particular bioactive material involved. A surfactant can
enhance solubility
of other formulation components to avoid aggregation or precipitation at
higher concentrations.
Surface active agents can, e.g., lower the surface tension of the suspension
or solution so that
bioactive materials are not denatured at gas-liquid interfaces, and/or so that
finer droplets can be
formed during spraying. The suspensions or solutions according to the
invention comprise
between about 0.001 and 5%; and preferably, between about 0.05 and 1%, or
about 0.2%, of a
nonionic surfactant, an ionic surfactant, or a combination thereof.
[045] Buffers can be added to the formulations of the invention, e.g., to
provide a suitable
stable pH to the formulations of the method and compositions of the invention.
Typical buffers
of the invention include, e.g., amino acids, potassium phosphate, sodium
phosphate, sodium
acetate, sodium citrate, histidine, glycine, sodium succinate, ammonium
bicarbonate, and/or a
carbonate. The buffers can be adjusted to the appropriate acid and salt forms
to provide, e.g., pH
stability in the range from about pH 3.0 to about pH 10.0, from about pH 4.0
to about pH 8Ø A
pH near neutral, such as, e.g., pH 7.2, is preferred for many compositions. In
one embodiment,
said VLP associated sugar glass comprises a phosphate buffer at pH 7.2 with
0.5 M NaCl.
[046] In another embodiment, said VLP and sugar glass composition is a powder
suspended in
a non-aqueous solvent that will not dissolve said sugar glass. In another
embodiment, said non-
aqueous solution is selected from the group consisting of triacetin, isoprppyl
myristate, medium
chain triglycerides, short, medium, and/or long-chain monoglycerides,
dimonoglycerides,
trimonoglycerides, aliphatic and aromatic alcohols, hydrofluoroether,
perfluoroether,
hyrofluoroamine, perfluoroamine, hydrofluorothioether, perfluorothioether, and
hydrofluoropolyether or a combination thereof (see WO 2005/099669, herein
incorporated by
reference in its entirety). In another embodiment, said powder is administered
to an animal
orally, via inhalation, intradermally, intranasally, intramusclarly,
intraperitoneally, intravenously,
or subcutaneously.
[047] In another embodiment, said powder is made by spray drying, milling or a
combination
thereof. In another embodiment, said powder has a mean particle diameter
between about 0.1
nm to about 100 microns. In another embodiment, said powder is suspended in a
non-aqueous
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solvent that will not dissolve said sugar glass. In another embodiment, said
powder is
administered to an animal orally, via inhalation, intradermally, intranasally,
intramusclarly,
intraperitoneally, intravenously, or subcutaneously. VLPs can have the protein
from a bacteria,
virus, fungal, parasite, as described above. In one embodiment, said VLP
comprises at least one
influenza protein. In another embodiment, said influenza protein is HA or NA.
In another
embodiment, said VLP comprises at least one RSV protein. In another
embodiment, said VLP
comprises a VZV protein. In another embodiment, said VZV protein is gE. In
another
embodiment, said composition further comprises an adjuvant.
[048] Another embodiment of the invention comprises a dry powder formulation
comprising a
VLP in a sugar glass. In one embodiment, said sugar glass is composed of a
monosaccharide or
disaccharide. In another embodiment, said monosaccharide is selected from the
group consisting
of glucose, sorbitol, galactose, mannose, and mannitol. In another embodiment,
said
disaccharide is selected from the group consisting of trehalose, maltose,
maltotriose, lactose,
lactulose, and sucrose. In another embodiment, said dry powder is made by
spray drying, freeze
drying, milling or a combination thereof. In another embodiment, said powder
has a mean
particle diameter between about 0.1 nm to about 100 microns. In another
embodiment, said
powder is administered to an animal orally, via inhalation, intradermally,
intranasally,
intramusclarly, intraperitoneally, intravenously, or subcutaneously. In
another embodiment, said
powder is reconstituted in a solvent before administration. In another
embodiment, said powder
is administered to an animal via inhalation using an inhaler and/or
subcutaneously using a jet of
high-pressure air. The composition may comprise any of the excipients
described above.
[049] Another embodiment of the invention comprises a method of delivering a
sugar glass
stabilized VLP comprising reconstituting a solid form of sugar glassified VLPs
in a solvent and
administering said reconstituted VLPs into an animal. In one embodiment, said
VLPs are
administered to an animal orally, via inhalation, intradermally, intranasally,
intramusclarly,
intraperitoneally, intravenously, or subcutaneously.
[050] Another embodiment of the invention comprises a method of delivering a
sugar glass
stabilized VLP comprising administering a solid form of sugar glassified VLPs
via inhalation
and/or injection. In one embodiment, said injection is administered via a jet
of high-pressure air.
[051] Another embodiment of the invention comprises a method of enhancing
thermal stability
of a VLP, comprising formulating said VLP into a sugar glass. In one
embodiment, said sugar
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glass is composed of a monosaccharide and/or disaccharide. In another
embodiment, said
monosaccharide is selected from the group consisting of glucose, sorbitol,
galactose, mannose,
and mannitol. In another embodiment, said disaccharide is selected from the
group consisting of
trehalose, maltose, maltotriose, lactose, lactulose, and sucrose. In another
embodiment, said
sugar glass comprising the VLP is a powder. In another embodiment, said powder
is made by
spray drying, freeze drying, milling or a combination thereof. In another
embodiment, said
powder has a mean particle diameter between about 0.1 nm to about 100 microns.
In another
embodiment, said powder is suspended in an organic solvent that will not
dissolve said sugar
glass. In one embodiment, said VLP comprises at least one influenza protein.
In another
embodiment, said influenza protein is HA or NA. In another embodiment, said
VLP comprises
at least one RSV protein. In another embodiment, said VLP comprises a VZV
protein.
[052] As also well known in the art, the immunogenicity of a particular
composition can be
enhanced by the use of non-specific stimulators of the immune response, known
as adjuvants.
Adjuvants have been used experimentally to promote a generalized increase in
immunity against
unknown antigens (e.g., U.S. Pat. No. 4,877,611). Immunization protocols have
used adjuvants
to stimulate responses for many years, and as such, adjuvants are well known
to one of ordinary
skill in the art. Some adjuvants affect the way in which antigens are
presented. For example, the
immune response is increased when protein antigens are precipitated by alum.
Emulsification of
antigens also prolongs the duration of antigen presentation. The inclusion of
any adjuvant
described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients
(2nd Edition),"
herein incorporated by reference in its entirety for all purposes, is
envisioned within the scope of
this invention. In one embodiment said VLP complexed with a sugar glass
formulation
comprises at least one adjuvant.
[053] Exemplary, adjuvants include complete Freund's adjuvant (a non-specific
stimulator of
the immune response containing killed Mycobacterium tuberculosis), incomplete
Freund's
adjuvants and aluminum hydroxide adjuvant. Other adjuvants comprise GMCSP,
BCG,
aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE),
lipid
A, and monophosphoryl lipid A (MPL). RIBI, which contains three components
extracted from
bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%
squalene/Tween 80 emulsion also is contemplated. MF-59, Novasomes , MHC
antigens may
also be used.
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[054] In one embodiment of the invention the adjuvant is a paucilamellar lipid
vesicle having
about two to ten bilayers arranged in the form of substantially spherical
shells separated by
aqueous layers surrounding a large amorphous central cavity free of lipid
bilayers. Paucilamellar
lipid vesicles may act to stimulate the immune response several ways, as non-
specific
stimulators, as carriers for the antigen, as carriers of additional adjuvants,
and combinations
thereof. Paucilamellar lipid vesicles act as non-specific immune stimulators
when, for example,
a vaccine is prepared by intermixing the antigen with the preformed vesicles
such that the
antigen remains extracellular to the vesicles. By encapsulating an antigen
within the central
cavity of the vesicle, the vesicle acts both as an immune stimulator and a
carrier for the antigen.
In another embodiment, the vesicles are primarily made of nonphospholipid
vesicles. In other
embodiment, the vesicles are Novasomes. Novasomes are paucilamellar
nonphospholipid
vesicles ranging from about 100 nm to about 500 nm. They comprise Brij 72,
cholesterol, oleic
acid and squalene. Novasomes have been shown to be an effective adjuvant for
influenza
antigens (see, U.S. Patents 5,629,021, 6,387,373, and 4,911,928, herein
incorporated by
reference in their entireties for all purposes).
[055] In one aspect, an adjuvant effect is achieved by use of an agent, such
as alum, used in
about 0.05 to about 0.1% solution in phosphate buffered saline. Alternatively,
the VLPs can be
made as an admixture with synthetic polymers of sugars (Carbopol(x) used as an
about 0.25%
solution. Some adjuvants, for example, certain organic molecules obtained from
bacteria;, act on
the host rather than on the antigen. An example is muramyl dipeptide (N-
acetylmuramyl-L-
alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan. In other embodiments,
hemocyanins
and hemoerythrins may also be used with VLPs of the invention. The use of
hemocyanin from
keyhole limpet (KLH) is preferred in certain embodiments, although other
molluscan and
arthropod hemocyanins and hemoerythrins may be employed.
[056] Various polysaccharide adjuvants may also be used. For example, the use
of various
pneumococcal polysaccharide adjuvants on the antibody responses of mice has
been described
(Yin et al., 1989). The doses that produce optimal responses, or that
otherwise do not produce
suppression, should be employed as indicated (Yin et al., 1989). Polyamine
varieties of
polysaccharides are particularly preferred, such as chitin and chitosan,
including deacetylated
chitin. In another embodiment, a lipophilic disaccharide-tripeptide derivative
of muramyl
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dipeptide which is described for use in artificial liposomes formed from
phosphatidyl choline
and phosphatidyl glycerol.
[057] Amphipathic and surface active agents, e.g., saponin and derivatives
such as QS21
(Cambridge Biotech), form yet another group of adjuvants for use with the VLPs
of the
invention. Nonionic block copolymer surfactants (Rabinovich et al., 1994) may
also be
employed. Oligonucleotides are another useful group of adjuvants (Yamamoto et
al., 1988).
Quil A and lentinen are other adjuvants that may be used in certain
embodiments of the present
invention.
[058] Another group of adjuvants are the detoxified endotoxins, such as the
refined detoxified
endotoxin of U.S. Pat. No. 4,866,034. These refined detoxified endotoxins are
effective in
producing adjuvant responses in vertebrates. Of course, the detoxified
endotoxins may be
combined with other adjuvants to prepare multi-adjuvant formulation. For
example,
combination of detoxified endotoxins with trehalose dimycolate is particularly
contemplated, as
described in U.S. Pat. No. 4,435,386. Combinations of detoxified endotoxins
with trehalose
dimycolate and endotoxic glycolipids is also contemplated (U.S. Pat. No.
4,505,899), as is
combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and
trehalose
dimycolate, as described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900.
Combinations of
just CWS and trehalose dimycolate, without detoxified endotoxins, is also
envisioned to be
useful, as described in U.S. Pat. No. 4,520,019.
[059] Those of skill in the art will know the different kinds of adjuvants
that can be conjugated
to vaccines in accordance with this invention and these include alkyl
lysophosphilipids (ALP);
BCG; and biotin (including biotinylated derivatives) among others. Certain
adjuvants
particularly contemplated for use are the teichoic acids from Gram-cells.
These include the
lipoteichoic acids (LTA), ribitol teichoic acids (RTA) and glycerol teichoic
acid (GTA). Active
forms of their synthetic counterparts may also be employed in connection with
the invention
(Takada et al., 1995).
[060] Various adjuvants, even those that are not commonly used in humans, may
still be
employed in other vertebrates, where, for example, one desires to raise
antibodies or to
subsequently obtain activated T cells. The toxicity or other adverse effects
that may result from
either the adjuvant or the cells, e.g., as may occur using non-irradiated
tumor cells, is irrelevant
in such circumstances.
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[061] Another method of inducing an immune response can be accomplished by
formulating
the VLPs of the invention with "immune stimulators." These are the body's own
chemical
messengers (cytokines) to increase the immune system's response. Immune
stimulators include,
but not limited to, various cytokines, lymphokines and chemokines with
immunostimulatory,
immunopotentiating, and pro-inflammatory activities, such as interleukins
(e.g., IL-1, IL-2, IL-3,
IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony
stimulating
factor (CSF)); and other immunostimulatory molecules, such as macrophage
inflammatory
factor, F1t3 ligand, B7.1; B7.2, etc. The immunostimulatory molecules can be
administered in
the same formulation as the influenza VLPs, or can be administered separately.
Either the
protein or an expression vector encoding the protein can be administered to
produce an
immunostimulatory effect.
[062] Where it is appropriate, the VLP associated with sugar glass of the
invention can be
administered, e.g., to a mammal. Said VLP and sugar glass composition of the
invention can
include, influenza, HIV, RSV, VZV VLPs.
[063] Said VLP and sugar glass composition can be administered to a patient by
topical
application. For example, the powder particles can be mixed directly into a
salve, carrier
ointment, pressurized liquid, gaseous propellants, and/or penetrant, for
application to the skin of
a patient. Alternately, the powder particles can, e.g., be reconstituted in an
aqueous solvent
before admixture with other ingredients before application.
[064] Said VLPs and sugar glass composition can be administered by inhalation.
Dry powder
particles less than about 10 um in aerodynamic diameter can be inhaled into
the lungs for
pulmonary administration. Optionally, powder particles of about 20 um, or
greater, in
aerodynamic diameter can be administered intranasally, or to the upper
respiratory tract, where
they are removed from the air stream by inertial impact onto the mucus
membranes of the
patient. The powder particles can alternately be reconstituted to a suspension
or solution for
inhalation administration as an aqueous mist.
[065] Said VLP and sugar glass composition can be administered by injection.
The powder
particles can be administered directly under the skin of a patient using,
e.g., a jet of high pressure
air. More commonly, the powder particles can be, e.g., reconstituted with a
sterile aqueous
buffer for injection through a hollow syringe needle. Such injections can be,
e.g., intramuscular,
intra venous, subcutaneous, intrathecal, intraperitoneal, and the like, as
appropriate. Powder
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particles of the invention can be reconstituted to a solution or suspension
with a bioactive
material concentration, e.g., from less than about 0.1 ng/ml to from less than
about 1 mg/ml to
about 500 mg/ml, or from about 5 mg/ml to about 400 mg/ml, as appropriate to
the dosage and
handling considerations. Reconstituted powder particles can be further
diluted, e.g., for multiple
vaccinations, administration through IV infusion, and the like.
[066] The appropriate dosage ("therapeutically effective amount") of the VLPs
material will
depend, for example, on the condition to be treated, the severity and course
of the condition,
whether the biologically active material is administered for preventive or
therapeutic purposes,
previous therapy, the patient's clinical history and response to the
biologically active material,
the type of biologically active material used, and the discretion of the
attending physician. The
VLPs are suitably administered to the patent once, or over a series of
administrations, and may
be administered to the patient at any time. The VLPs may be administered as
the sole treatment
or in conjunction with other drugs or therapies useful in treating the
condition in question.
[067] As a general proposition, the therapeutically effective amount of VLPs
administered will
be in the range of about 0.00001 to about 50 mg/kg of patent body weight
whether by one or
more administrations, with the typical range of protein used being from less
than about 0.01
ng/kg to about 20 mg/kg, more preferably about 0.1 mg/kg to about 15 mg/kg,
administered
daily, for example (as measured by the antigenic protein on said VLP).
However, other dosage
regimens may be useful. The progress of this therapy is easily monitored by
conventional
techniques.
[068] The invention also encompasses methods of increasing the "shelf-life" or
storage stability
of VLPs stored at elevated temperatures. Increased storage stability can be
determined by
recovery of biological activity in accelerated aging trials. The dry particle
compositions
produced by methods of the invention can be stored at any suitable
temperature. Preferably, the
compositions are stored at about 0 C. to about 80 C. More preferably, the
compositions are
stored at about 20 C. to about 60 C. Most preferably, the compositions are
stored at ambient
temperatures.
EXAMPLES
Example 1 Freeze-drying of VLPs
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[069] H5N1 Clade 2 Influenza VLPs were made and purified according to the
methods
described in co-pending application 11/582,540, filed October 18, 2006, herein
incorporated by
reference in its entirety. The VLPs were purified and suspending in phosphate
buffered saline,
pH 7.2 with the desired concentration of NaCl in a PETG bottle with the
desired concentration
of sugar. The formulations are described in Table 1.
Table 1 Formulations
Formulation Vehicle Sugar concentration
code m /mL
Control (C) PB, pH 7.2 with 0.5M NaCl None
C o-I PB, pH 7.2 with 0.5M NaCl Trehalose (60 m /mL
C o-II PB, pH 7.2 with 0.15M NaCl Trehalose (60 m /mL
C o-III Water with 0.01 % w/v PS80 Trehalose (60 m /mL
C o-IV PB, pH 7.2 with 0.15M NaCl Mannitol (50 m /mL
Cryo-V PB, pH 7.2 with 0.15M NaCl Trehalose (30 mg/mL) +
Mannitol (25 m /mL
Cryo-VI PB, pH 7.2 with 0.15M NaCl Sucrose (30 mg/mL) +
Mannitol (30 m /mL
[070] Next, 1.2 mL of the formulation (aqueous suspension) was aliquoted into
2 mL USP
Type I glass vials. The potency of the formulations was in the range of 40-80
gg HA/mL. The
potency was measured using the quantitative single radial immunodiffusion
(SRID) assay. Lyo-
stoppers (13 mm, grey chlorobutyl) were placed on the vials and all the
formulations were
subjected to glassification via freeze-drying using a 2 sq.ft. Hull model
2FS48C freeze dryer
according to the parameters outlined in the Table 2.
Table 2 Freeze Drying Parameters
Shelf Temperature Soak Time Ramping Pressure Set Point
Step Setpoint ( C) (hours) Rate ( C/hour) (mHg)
Product Loading 5 2 5-10 inHg vac
30 5-10 inHg vac
Freeze/Evacuation -50 3 5-10 inHg vac
30 80
-30 1 80
Primary Drying, Step 1
30 80
-25 45 80
Primary Drying, Step 2
15 80
Secondary Drying 25 8 80
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Stoppering Pressure 14.7 psi
Total Processing Time 66 hrs
[071] After glassification the material in each vial was reconstituted with
1.2 mL of sterile
water for injections (WFI) before analyzing VLP potency and stability (post-
lyophilization).
Example 2 VLP analysis after glassification
[072] After resuspention, Single Radial Immunodiffusion (SRID), total protein
analysis,
Particle size analysis, HA and NA activity were checked. Total protein was
measured by the
BCA method (using a commercial kit that employs bicinchoninic acid). Particle
size was
measured using the Malvern zeta sizer (dynamic light scattering principle). HA
activity was
measured using haemagglutination assay employing Turkey RBCs and NA activity
was
measured by fluorimetry using Munana reagent). In addition, SDS PAGE and
western blot
analysis were conducted. Tables 3 to Table 7 and Figure 1 summarize the
results of these
analysis.
TABLE 3 SRID (pg HA/mL
Before FD After FD % change
Control 71.72 59.19 -17.47
Cryo-l 71.98 56.88 -20.98
C o-II 68.94 70.01 1.55
C o-III 41.29 36.06 -12.67
Cryo-IV 66.34 54.71 -17.53
Cryo-V 67.01 69.58 3.84
C o-VI 66.99 64.78 -3.30
TABLE 4 Total proteins by BCA (mg/ L)
Before FD After FD % change
Control 0.13 0.133 -0.58
Cryo-l 0.15 0.143 -4.67
C o-II 0.13 0.134 0.59
C o-III 0.12 0.12 -1.66
Cryo-IV 0.12 0.122 -0.48
Cryo-V 0.14 0.131 -6.38
C o-VI 0.13 0.126 -6.59
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TABLE 5 Particle size nm
Before FD After FD % change
Control 156 274 75.64
Cryo-I 161 163 1.24
C o-II 155 158 1.94
C o-III 1320 1190 -9.85
Cryo-IV 155 176 13.55
Cryo-V 154 158 2.60
C o-VI 155 159 2.58
TABLE 6 HA titer
Before FD After FD % change
Control 2048 8192 300.00
C o-I 4096 4096 0.00
C o-II 4096 8192 100.00
C o-III 128 32 -75.00
Cryo-IV 256 8192 3100.00
Cryo-V 2048 8192 300.00
C o-VI 2048 2048 0.00
TABLE 7 NA activity (mU/mL)
Before FD After FD % change
Control 7 5.30 -24.29
C o-I 7.3 6.50 -10.96
C o-II 6.5 6.30 -3.08
C o-III 0.3 0.45 50.00
Cryo-IV 5.8 5.00 -13.79
Cryo-V 6 5.70 -5.00
C o-VI 6.4 5.80 -9.38
[073] Figures 1 A and B compares VLPs comprising HA and NA via SDS and western
blots
before glassification and after glassification. As shown, after glassification
of the VLPs there is
no difference in the amount HA and NA as shown in the gels. Thus,
glassification does not
reduce or chew up the VLPs or antigen expressed on the VLPs.
[074] The these results show that the formulations Control, Cryo-II and Cryo-V
in particular
retained the physical, chemical and biological properties of the VLPs most
effectively upon
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freeze-drying. It was surprising to see a protection effect without any sugar
(control). But the
benefit of sugar-glassification can be shown from the stability study at
higher temperatures.
Example 3 VLP stability at high temperatures
[075] The recommended storage temperature for an aqueous suspension containing
VLPs is 4-
8 C. The freeze-dried vials (control, cryo-II and cryo-V for 5 or 12 weeks)
where stored at 25 C
and 50 C to investigate the protection effect of sugars on VLP stability. The
VLPs were
analyzed using the same methods as described above. The results are summarized
in the
following tables (FD = freeze drying).
Table 8 SRID HA/mL)
weeks 5 weeks @ 12 weeks 12 weeks
Before FD After FD 25 C 50 C 25 C 50 C
Control 71.72 59.19 78.20 0.00 41.59 0.00
C o-II 68.94 70.01 80.60 33.38 83.27 59.99
Cryo-V 67.01 69.58 78.80 52.41 67.30 36.88
Table 9 Particle size (nm)
5 weeks 5 weeks @ 12 weeks 12 weeks
Before FD After FD 25 C 50 C 25 C 50 C
Control 156 274 364.00 414.00 359.9 386.4
C o-I I 155 158 160.00 178.00 160.4 166.1
Cryo-V 154 158 160.00 182.00 164.2 175.2
Table 10 HA titer
5 weeks 5 weeks @ 12 weeks 12 weeks
Before FD After FD 25 C 50 C 25 C 50 C
Control 2048 8192 1024 256 512 64
C o-II 4096 8192 2048 64 2048 2048
Cryo-V 2048 8192 2048 2048 2048 2048
[076] Even though there seem to be no significant differences in the data
between control and
sugar-glassified products at 25 C up to 5 weeks, there was a significant drop
in potency (SRID
value) at 12 weeks for control. At 50 C, the control formulation lost its
potency completely
within 5 weeks and showed aggregation and loss in bioactivity (as indicated by
HA titer).
[077] The sugar-glassified formulations (cryo-II and V) preserved the potency,
particle size and
HA titer at 25 C up to 3 months. At 50 C a potency loss of approx. 13% and 45%
was observed
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for cryo-II and cryo-V respectively while the particle size and HA titer
remained unaffected.
Trehalose appeared to preserve the integrity of the VLPs better than the
trehalose + mannitol
combination.
Example 4 Spray-drying of VLPs
[078] H5N1 Clade 2 Influenza VLPs were made and purified according to the
methods
described in co-pending application 11/582,540, filed October 18, 2006, herein
incorporated by
reference in its entirety.
[079] Placebo was prepare in 6% w/v solution of Trehalose by dissolving 24
grams of
Trehalose in 400 mL of phosphate buffered saline, pH 7.2 with 0.15 M NaCl. The
solution was
sterilized by passing it through a O.22 membrane filter.
[080] VLPs were collect in Phosphate buffered saline, pH 7.2 with 0.15 M NaCl
(potency of
about 45 gg HA/mL) in a PETG bottle. Trehalose was then added to constitute 6%
w/v solution.
The solution was mixed gently (hand shaking) until all Trehalose is dissolved.
[081] Next, the Spray-dryer was set-up and run under the following conditions:
SD-Micro
(Niro) with glass chambers, 2.4/3/4 mm Morprene tubing with "Y". Compressed
air was used as
the drying air. The conditions for each of the runs are in Table 11.
Table 11 S ay Dry Conditions
Run Inlet Outlet Atom Atom rate Airflow Spray
temp( C) temp( C) pressure (kg/h) (kg/h) rate
(bar) (g/min)
1 160 90 1.6 1.6 29.9 6.69
2 160 100 1.1 1.2 29.9 4.37
Run #1 = Placebo formulation
Run #2 = VLP-sugar formulation
[082] After resuspention, Single Radial Immunodiffusion (SRID), HA titer
particle size and
NAA were checked (as described above). In addition, SDS PAGE blot analysis was
conducted.
The results are summarized on Table 12 and on the gels in Figure 2.
Table 12 Summary of Spay Dried VLPs Assays
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Sample ID NAA HA SRID ( g/ml) Particle
(mU/ml) Titer Size (nm)
Before spray drying 48.0 1024 40.8 173.4
35.4
200mg dried powder in (354 gg HA/gm
2m1 WFI after spray drying 25.1 1024 dried product) 203.8
64.8
500mg dried powder in (259 gg HA/gm
2m1 WFI after spray drying 64.0 2048 dried product 221.9
Note: Theoretical potency (assuming 5% w/w residual moisture): 45 g HA per
115 mg dried product.
Practical potency: 45 g HA per 127 to 173 mg of dried product
[083] These data show that the practical potency ( the potency achieved after
spray drying in
terms of gg HA/mg of dried powder) was comparable to theoretical potency (the
potency that is
estimated by a calculation based on material inputs e.g. quantity of VLPs,
trehalose and an
assumption of approx. 5-10% moisture upon drying and greater than 95%
recovery), thus
indicating a preservation of original properties of the VLPs after spray-
drying. In addition, the
SDS-PAGE and Western blot gels (Figures IA) indicate the integrity of the
three principal
proteins (HA, NA and M1) with no band shifting or broadening for any of the
formulations,
before and after freeze-drying.
[084] All publications, patents and patent applications herein are
incorporated by reference to
the same extent as if each individual publication or patent application was
specifically and
individually indicated to be incorporated by reference.
[085] The foregoing detailed description has been given for clearness of
understanding only
and no unnecessary limitations should be understood therefrom as modifications
will be obvious
to those skilled in the art. It is not an admission that any of the
information provided herein is
prior art or relevant to the presently claimed inventions, or that any
publication specifically or
implicitly referenced is prior art.
[086] Unless defined otherwise, all technical and scientific terms used herein
have the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs.
[087] While the invention has been described in connection with specific
embodiments thereof,
it will be understood that it is capable of further modifications and this
application is intended to
cover any variations, uses, or adaptations of the invention following, in
general, the principles of
the invention and including such departures from the present disclosure as
come within known or
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customary practice within the art to which the invention pertains and as may
be applied to the
essential features hereinbefore set forth and as follows in the scope of the
appended claims.
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