Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITION AND METHOD FOR STABLE INJECTABLE LIQUIDS
Background of the Invention
Vaccines or drugs in solution ready for injection are inherently unstable and
need refrigeration. The pharmaceutical industry has traditionally tackled the
instability problem by freeze-drying drugs. This is expensive, inconvenient
and
inherently dangerous, since incorrect reconstitution of dried drugs can result
in wrong
doses or contaminated solutions. Many attempts have been made over the past
100
years to develop robust, stable, ready-to-inject liquid formulations with
pitiful lack
of success. Only inherently tough small molecule drugs can survive in aqueous
solution with a useful shelf life.
This problem is particularly acute in the vaccine industry. By the year 2005
it is estimated that 3.6 billion doses of vaccine will have to be administered
world-
wide. It has been stated by the World Health Organization (WHO) that this will
not
be possible using standard vaccine formats which need to be refrigerated at
all times
("Revolutionizing Immunizations." Jodar L., Aguado T., Lloyd J. and Lambert P-
H.
Genetic Engineering News Feb 151998). A "cold chain" of refrigerators is
currently
in use, which stretches from the vaccine factories to provincial towns in the
developing world. The cost of the cold chain for the vaccine industry and for
non¨
governmental health organizations running immunization campaigns is enormous.
The WHO has estimated that just the maintenance cost of the cold chain is over
SUS
200 million annually. In addition, immunization campaigns may reach only those
living.lose to the last link of the cold chain.
Vaccination campaigns require medically trained staff to ensure that the dose
is correctly injected and shows no signs o f degradation. The need to
reconstitute some
vaccines, such as measles, yellow fever and BCG, in the field is also a
serious
concern. This must be done precisely to ensure correct dosage and it also
introduces
a potential source of contamination, which has frequently led to clinical
disasters. In
addition, it is often necessary to give more than one vaccine at a session and
this may
require multiple injections, as particular mixtures or "multivalent" vaccines
may not
be available due to the chemical incompatibility of some of the components.
The
WHO has highlighted these problems by actively encouraging research into the
next
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generation of stable vaccines which have no need for refrigeration and which
need
no reconstitution ("Pre-Filled Monodose Injection Devices: A safety standard
for new
vaccines, or a revolution in the delivery of immunizations?" Lloyd J. and
Aguado
M.T. WHO publication May 1998. "General policy issues : injectable solid
vaccines
: a role in future immunization?" Aguado M. T., JOdar L., Lloyd J., Lambert P.
H.
WHO publication No A59781.)
An ideal solution to this problem would be completely stable, ready-to-inject
formulations. Such stable vaccines could be packed as individual doses in an
injecting device itself, or, for mass immunization campaigns, shipped in
larger
volumes and administered by means of a needle-free jet injector. The
transdermal
delivery of dry solids by gas jet injection has been described (Sarphie DF,
Burkoth
TL. Method for providing dense particle compositions for use in transdermal
particle delivery. PCT Pub No. WO 9748485 (1996)) and transdermal vaccination
with dry DNA vaccines is apparently very effective ("PowderJect's Hepatitis B
DNA
Vaccine First To Successfully Elicit Protective Immune Response In Humans" at
http://www.powderj ect. corn/pressre I eases. h tm (1998)).
The hypersonic shockwave of helium gas that is used to drive these powder
injectors has a limited power and cannot deliver its dose of fine particles
intra-
muscularly. This is because the low-mass particles cannot achieve adequate
momentum for deep penetration. While the intradermal delivery of DNA vaccines
coated on to colloidal gold particles is adequate for good immunogenicity, the
common vaccines, adjuvanted with insoluble aluminum or calcium salts, induce
unacceptable skin irritation. They must be given intramuscularly. What is
required
is a flexible system capable of a range of delivery depths, from intradermal
to deep
intramuscular, similar to that achievable by existing needle and syringe
technology.
For mass vaccination campaigns this has been solved by the development of the
liquid jet injector capable of accelerating a narrow (-0.15 mm diameter)
stream of =
liquid, using pressures of around 3,000 psi, into a "liquid nail". This device
delivers
its dose painlessly through the skin into the deep subcutaneous or muscle
tissue by
punching a minute hole through the epidermis. The high momentum imparted to
the
liquid stream ensures deep penetration. To date, the injected drugs and
vaccines have
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been water-based but because of the instability problems discussed above, the
range
of stable aqueous products accessible to this technology is very limited.
It is now recognized that a wide range of bioactive molecules may be
stabilized by drying in sugar glasses (Roser B. "Protection of proteins and
the like"
UK patent No 2,187,191. Roser B and Colaco C. "Stabilization of biological
macro-
molecular substances and other organic compounds" PCT Pub No WO 91/18091.
Roser B. and Sen S. "New stabilizing glasses". PCT patent Application no:
9805699.7. 1998). These dry, stabilized actives are unaffected by hostile
environments such as high temperatures and ionizing radiation.
The mechanism underlying the remarkable stabilization of molecules by
sugars is glass-transformation. 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
crystallization
is a crucial property of a good stabilizer. Trehalose is good at this (Green
JL. &
Angel CA. Phase relations and vitrification in saccharide water solutions and
the
trehalose anomaly J. Phys. Chem. 93 2880-2882 (1989)) but not unique. Further
drying progressively solidifies the syrup, which turns into a glass at a 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.
In this
form the molecules can remain unchanged providing one other condition is met.
This
is the second crucial property of a good stabilizer viz, that it is chemically
inert and
non-reactive. Many glasses fail because they react with the product on
storage.
Obvious problems occur with reducing sugars, which may form good physical
glasses
but then their aldehyde groups attack amino groups on the products in a
typical
Maillard reaction. This is the main reason that many freeze-dried
pharmaceuticals
require refrigerated storage. Non-reactive sugars give stable products, which
require
no refrigeration at all.
Biomolecules immobilized in sugar glass are also stable in non-aqueous
industrial solvents in which they themselves and the sugar are both insoluble
(Cleland
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JL. and Jones AJS. "Excipient stabilization of polypeptides treated with
organic
solvents" US Patent No.5,589,167. (1994)). Since the sugar glass acts as an
impermeable barrier in a non-solvent liquid, the biomolecules in solid
solution in the
glass are protected both from the chemical reactivity of the solvent and from
the
environment. Providing the liquid itself is stable, sensitive products in
suspended
glass particles constitute a stable two phase liquid formulation. Industrial
solvents
of the kind described by Cleland and Jones (1994) have a limited utility in
processing. Substituting a bio-compatible non-aqueous liquid would enable
stable
liquid formulations of even the most unstable drugs, vaccines and diagnostics
to be
formulated.
The first generation of stable non-aqueous liquids designed to be used in drug
or vaccine delivery (B.J. Roser and S.D. Sen "Stable particle in liquid
formulations".
PCT Patent Application no. GB98 / 00817 described formulations of powders of
stabilizing glasses containing the active, suspended in injectable oils such
as sesame,
arachis or soya oil or simple esters such as ethyl oleate. The suspended sugar
glass
particles are of an intensely hydrophilic nature while the oils are
hydrophobic.
Because of the strong tendency of the hydrophilic and hydrophobic phases to
separate, the sugar glass particles tended to clump together. In order to
stabilize such
"water in oil" type suspensions the use of oil-soluble surfactants dissolved
in the
continuous oil phase was often required.
These low 111,B (Hydrophilic / Lipophilic Balance) surfactants accumulate at
the interface between the hydrophilic particles and the oil and coat them with
an
amphiphilic layer which is more compatible with the continuous oil phase.
Because
each sugar glass particle is separated from its neighbors by dry oil, no
chemical
interaction can go on between particles. It is therefore possible to have
several
different populations of particles, each containing a different potentially
interactive
molecule, in the same oil preparation, without them being able to interact.
Complex
multivalent vaccines can be produced in this way.
However, this approach has been subsequently found to have certain
drawbacks that prevent it from being a universal solution. These include the
inevitable sedimentation of the suspended particles, which have a typical
density
CA 02689856 2010-01-13
around 1.5 g / cm', in the less dense, oily vehicle. The patent acknowledges
this
problem and aims to solve it by reducing the particle size to below 1 ,um in
diameter
in order for them to remain suspended by thermodynamic forces such as Brownian
motion. The requirement for all particles to be below 1 ,um in diameter is a
5
disadvantage of the proposed formulations. Achieving such small particle
powders
is by no means an easy task. Improved spray drier designs may be able to
achieve this
but the small particle size would prevent the use of cyclone type collectors
and
require a system of filters for product recovery.
Reducing particles to sub-micron size may also, in theory, be achieved after
the particles are suspended in the oil, with high-pressure micro-homogenizing
equipment such as the Microfluidizer (Constant Systems Inc.). This involves an
extra
step to the process and we have found it not to be very efficient in breaking
down
spray-dried sugar glass microspheres, which have very high mechanical strength
because of their spherical shape. This mandates multiple passes through the
equipment. Even then, this tends to leave a number of the larger particles
untouched
and therefore would require a subsequent filtration or sedimentation step to
remove
them. Also, the high viscosity of the suspensions in the usual oily vehicles
makes
=
them difficult both to draw up into the syringe and requires that they be
injected
slowly. It precludes fast flows through fine nozzles such as are experienced
in a
liquid jet injector system.
It has also been found that particles suspended in an oil, specially when
containing a low HLB surfactant, are difficult to extract subsequently into an
aqueous
environment because, surprisingly, they maintain a tightly bound, water
repellent coat
of oil around them, even after washing in aqueous buffer. They therefore
require very
vigorous shaking and mixing or the addition of yet more water-soluble
detergent (this
time with a high HLB) for the particles to leave the oil phase and enter the
water
phase. This becomes more of a problem as the particle size is reduced. The
final
outcome is often a rather messy mixed emulsion rather than two cleanly
separate
phases. In the body this problem can cause slow and unpredictable release of
the
active rather than the prompt and predictable delivery required. Extraction in
vitro
into an aqueous environment results in the oil floating on top of the aqueous
phase
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containing the dissolved active. This may not be acceptable for certain in
vitro
applications such as diagnostic kits or automated assay systems. Finally, most
of the
natural, FDA-approved, oils, which can be used clinically, are vulnerable to
photodegradation, oxidation or other forms of damage and require careful
storage in
the dark at relatively low temperatures. Additionally, they are not completely
chemically inert so that they can slowly react with the suspended particles.
The Alliance Pharmaceutical Company has explored the use of powders of
water-soluble substances in the remarkable new non-aqueous perfluorocarbon
liquids
(Kirkland WD Composition and method for delivering active agents. US Patent No
5,770,181. (1995)). This patent is primarily concerned with the function of
the PFCs
as oral contrast enhancing agents for diagnostic imaging of the intestines.
The water-
soluble powders exemplified therein were added to improve the palatability or
the
enhancement of the contrast effect in the gastrointestinal tract of the PFCs.
However,
Kirkland perceptively realized that these liquids could also be used for drug
delivery
although there are no examples given. In particular, only shelf stable
commercially
available powders are exemplified in the patent. We have now found that
fragile
actives stabilized in sugar glass microspheres can be engineered to produce
extremely
stable two-phase PFC liquid formulations for both oral and parenteral
delivery. This
greatly extends the utility of the Kirkland patent to the delivery of
parenteral drugs
and vaccines in ready-to-inject formulations that require no refrigeration of
any kind.
Of particular value is the discovery that the low viscosity, high density and
low
surface tension of PFCs means that these stable suspensions can be delivered
by
automatic devices such as liquid jet injectors. This opens up two important
additional
fields to this technology namely mass immunization campaigns and also self
injection.
Perfluorocarbons (PFCs) are novel, extremely stable liquids produced by the
complete fluorination of certain organic compounds. They cannot be classified
as
either hydrophilic or lipophilic, as they are in fact essentially immiscible
with both
oil and water or any other solvent whether polar or non-polar, except other
PFCs.
(Reviewed in Krafft MP & Riess JG. "Highly fluorinated amphiphiles and
colloidal
systems, and their applications in the biomedical field. A contribution."
Biochimie
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80 489-514 1998). Furthermore, they do not participate in hydrophobic
interactions
with oils nor hydrophilic interactions with water or hydrophilic materials. As
a
consequence gross phase separation, as seen when hydrophilic particles clump
strongly together in oil, tends not to occur in PFCs. They may not require
surfactants
to produce stable suspensions, but fluorohydrocarbon (FHC) surfactants are
available
(Krafft & Riess 1998) and are active at minute concentrations in PFC liquids.
At
these very low concentrations FHC surfactants can ensure perfect monodisperse
systems of certain particles which show a tendency to aggregate in their
absence. The
PFC liquids themselves are chemically completely non-reactive and the lower
molecular weight types do not accumulate in the body but, being volatile, are
eventually exhaled in the breath.
Because they are excellent solvents for gases, PFCs have already been used
in large quantities in very special clinical applications. Their ability to
exchange
carbon dioxide for dissolved oxygen is better than that of haemoglobin. This
was first
demonstrated in "bloodless rats" by R.P. Geyer in 1968 (Geyer RP, Monroe RG &
Taylor K. "Survival of rats totally perfused with perfluorocarbon-detergent
preparation." in: Organ Perfusion and Preservation, J.V Norman, J Follunan,
L.E.
Hardison, L.E Ridolf and F.J. Veith eds. Appleton-Century-Crofts, New York..
85-
95 (1968)). Perfluorooctyl bromide, in the form of a PFC-in-water emulsion and
under the trade name Oxygentmi (Alliance Pharmaceutical Corp.) is presently
being
evaluated in humans as an alternative to blood transfusion for certain
surgical
procedures. PFCs have also been used by inhalation, as liquids, into the lungs
as a
treatment for respiratory distress syndrome in premature babies.
Their high density combined with chemical inertness has also been found to
be valuable. Perfluorophenanthrene, under the trade name Vitreonmf (Vitrophage
Inc.), is used to prevent collapse of the capsule of the eye during surgery
and to
permit repositioning of detached retinas. PFCs have also been used as contrast
media
for Magnetic Resonance Imaging (MRI) and for this purpose it has been reported
that
hydrophilic powders may be suspended in them in order to either improve their
imaging properties or make them more palatable.. (Kirkland W.D. "Composition
and
method for delivering active agents" US patent 5,770,181. 1998). This patent
also
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suggests the use of PFCs as the continuous phase for delivering particulate
water-
soluble drugs. Since the number of parenteral drugs, which are stable as dry
powders
at room temperature is limited, this patent does not have applicability to the
majority
of injectable drugs. However, the combination of drug stabilization in
microsphere
powders of sugar glasses as described in Roser and Garcia de Castro (1998) and
injectable PFCs renders this technology applicable to virtually all parenteral
drugs
and vaccines.
Summary of the Invention
The invention herein uses a two-phase system, with PFCs as the continuous
phase containing a discontinuous glass phases in suspension, as drug delivery
preparations. Perfluorocarbon based preparations present major advantages in
that
different PFCs may be blended to obtain final mixtures with densities ranging
from
approximately 1.5 to 2.5 g / cm3. This allows for the particles to be
formulated with
densities matching the suspension fluid in order that they do not float or
sink to the
bottom of the container but remain in the form of a stable suspension.
Particles
therefore need not be of submicron size as required in oil based preparations
to
prevent sedimentation, but may vary greatly in size. The ultimate particle
diameter
is governed only by the purpose of the preparation. Preparations intended for
needle
injection or jet injection could contain particles in the range of 0.1 to 100
micrometers, or preferably 1 to 10 micrometers. This allows for a great
simplification
in the manner of manufacture of the particles and avoids the necessity for
extremely
small particle size production by milling. Particles can be made by
conventional
spray drying or by freeze-drying followed by simple dry or wet milling. When a
high
solids content in the suspension is needed it is desirable that the particles
be spherical
in shape. Irregularly shaped particles have a much greater tendency to "bind"
together inhibiting free-flow, while spherical particles have an inherent
"lubricity"
enabling solids contents of well over 20% to be achieved. Such particles are
easily
made by spray-drying, spray-freeze-drying or emulsion solidification.
The suspended powders, if formulated appropriately, need no surfactants,
producing stable suspensions from which the sugar glass particles dissolve
almost
instantly when shaken with water. If minor aggregation is perceived as a
problem,
CA 02689856 2010-01-13
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small amounts of a FHC surfactant such as described in Krafft and Riess (1998)
may
be advantageously added to the PFC fluid either before or after the admixture
of the
stable powder. Like the PFCs, these FHCs are inherently extremely inert and
non-
reactive. There is thus no solvation of the particles and no chemical reaction
between
the suspended particles and the PFC phase. Because both the sugar glass
particles
and the PFC liquid are environmentally stable there is no degradation due to
light,
high temperatures, oxygen etc. They have negligible in vivo or in vitro
toxicity and
they have been extensively tested and approved by the regulatory authorities
by being
infused into both animals and man in large volumes for blood replacement
purposes.
While high molecular weight PFCs have been reported to accumulate. in the
liver, the
lower molecular weight examples used in this application are eventually
eliminated
from the body in the exhaled breath.
Their low surface tension and their low viscosity enables them to flow very
easily through the narrow bores which may be encountered in hypodermic
needles,
automated systems or liquid jet injectors. PFCs are excellent electrical
insulators and
therefore it is easy to achieve monodisperse suspensions of particles carrying
the
same small surface electrostatic charge. They are dry and completely non-
hygroscopic liquids. Their very low water content maintains the dryness of the
suspended powders, preventing the dissolution or degradation of the
incorporated
actives. Their unique lack of solvent properties make them ideal for
suspending
either hydrophilic or hydrophobic particles and means that the final
suspensions are
compatible with virtually any materials used.in containers or delivery
devices. This
is in contrast with oil-based preparations, which can cause severe jamming of
syringes for example, by swelling the rubber seals on the plungers. PFCs can
be
obtained in a range of densities, vapor pressures and volatilities, (Table I).
Their high
densities cause them to sink in most conventional buffers, allowing for easy
separation from the product particles, which dissolve in the aqueous phase
that floats
on top. This therefore facilitates their use for in vitro applications such as
diagnostics.
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In one aspect of the invention, there is provided a pharmaceutical composition
comprising an active ingredient carried by particles of sugar glass having a
diameter in
the range of 0.1 to 100 micrometres, the particles being suspended in a
biocompatible
perfluorocarbon liquid, wherein the densities of the particles and
biocompatible liquid are
5 matched such that the particles remain in suspension.
Detailed Description of the Invention
Table I Properties of Some PFCs
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Pertluoro- MW Density Viscosity Surface tension Vapor pressur
(Kg/L) (mPas) (mN/m) (mbar)
hexane 338 1.682 0.656 11.1 294
-n-octane 438 1.75 1.27 16.98 52
decalin 462 1.917 5.10 17.6 8.8
phenanthrene 624 2.03 28.4 19 <1
The use of PFCs as vehicles for delivery of pharmacological agents or
bioactive
agents was previously suggested in Kirkland (1995). This patent exemplified
only
inherently stable commercially available flavoring or effervescent powders and
the like.
It contained no examples of any stabilized bioactives such as vaccines or
pharmaceuticals. Furthermore it does not consider the possibility of making an
injectable (parenteral) preparation by using PFCs as the suspension vehicle
for the
active particles. In order to achieve a stable formulation of inherently
fragile
biomolecules with a long shelf life using PFCs as the non-aqueous vehicle, the
particles
would preferably be formulated to contain a glass-forming agent capable of
stabilizing
the incorporated active. This may be from a variety of sugars, including
trehalose,
lactitol, palatinit, etc as described in PCT No. WO 91/18091 or more
preferably other
more effective monosaccharide sugar alcohols or glass forming agents as
described in
UK Pat. Application no. 9820689.9 (priority document for EP 1071465).
In order to prevent the particles from floating in the dense PFC phase, it is
advantageous to incorporate a density-regulating agent in the particles. This
may be
either a soluble salt such as sodium or potassium chloride or sulphate or more
preferably, an insoluble material such as barium sulphate, calcium phosphate
titanium
dioxide or aluminum hydroxide. The insoluble, non-toxic materials are
preferred since
the release of large amounts of ionic salts in the body can cause considerable
local pain
and irritation. The insoluble materials may, in some cases, such as in vaccine
preparations, be part of the active preparation as an adjuvant. The density
regulator may
be in solid solution in the sugar glass particles or an insoluble particulate
material in
suspension in the sugar glass. When correctly formulated, the sugar glass
particles are
approximately density matched with the PFC liquid, are
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buoyancy neutral, and neither float nor settle but remain in stable suspension
without
caking.
Because PFC liquids are good electrical insulators, with a typical resistivity
of greater than 10" ohm.cm, tiny surface charges on the suspended particles
can have
significant effects on suspension stability. In order to prevent the suspended
particles
from aggregation due to weak short-range forces, they are preferably
manufactured
containing an excipient such as lysine or aspartic acid capable of donating a
weak
residual electrostatic charge to the dry particles. This prevents aggregation
by
ensuring charge repulsion of the particles, similar to that seen in stable
colloids.
Alternatively, small amounts of FHC surfactants such as perfluorodecanoic acid
may
be advantageously dissolved in the PFCs to give dispersed, preferably
monodisperse,
suspensions.
These particles may be manufactured in a number of ways, including air,
spray or freeze-drying and need not to be particularly small but may be a
heterogeneous mix of sizes ranging between 0.1 and 100t in diameter. For some
applications even millimeter-sized particles may be suitable.
The use of these stable suspensions is restricted to neither parenteral use as
exemplified above nor oral use as exemplified in US 5,770,181. Because the PFC
liquid vehicle is so non-toxic and non-reactive, it is an ideal vehicle for
mucosa!,
including intrapulmonary, intranasal, intraocular, intra rectal and
intravaginal
delivery. The ability, provided by this patent, to produce stable, sterile and
non-
irritant formulations for mucosal delivery of even very unstable drugs or
vaccines is
a considerable advance. Also, the very dry and completely non-hygroscopic
nature
of the PFC liquid greatly assists in the maintenance of sterility of these
preparations
during prolonged storage and intermittent use as micro-organisms cannot grow
in the
absence of water
Since volatile perfluorohydrocarbons and chlorofluorocarbons have long been
used as propellants in inhalers designed to achieve drug delivery to the deep
lung, the
stable PFC formulations described herein are ideal for generating fine mists
of liquid
STASIS droplets for intrapulmonary delivery. For this application, the size of
the
particles which constitute the discontinuous suspended phase in the PFC
droplets is
CA 02689856 2010-01-13
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important and should not exceed 1 to 5 gm, preferably 0.1 to 1 um in diameter.
For
delivery to other mucosal surfaces in the nose or eye, the particle size is
less
important and can be up to 100 gm or even several mm in diameter.
Description of the Drawings
Figure 1
Alkaline phosphatase (Sigma Aldrich Ltd.) was stabilized in a glass based on
mannitol 33.3%, calcium lactate 33.3% and degraded gelatin 33.3% (Byco C,
Croda
Colloids Ltd.), spray dried as microspheres and stored at 55 C either as dry
powder
or as a stable suspension in Perfluorodecalin. The activity remained around
the 100%
mark (103% at 20d and 94% at 30d). There was more loss in the dry powder which
was not suspended in PFC (around 80% of activity remained)
Figure 2
A commercial tetanus toxoid vaccine, (#T022 kindly supplied by Evans
Medeva plc) was formulated as a density-matched powder using added calcium
phosphate in 20% trehalose solution. It was freeze-dried by spraying into
liquid
nitrogen using a two fluid nozzle followed by freeze drying the frozen
microsphere
powder in a Labconco freeze dryer with the initial shelf temperature at ¨40 C
throughout primary drying. The antibody response of six group of 10 Guinea
Pigs
was measured 4, 8 and 12 weeks after being injected with the same dose of
ASSIST
stabilized Tetanus Toxoid vaccine reconstituted in saline buffer or as
anhydrous
preparations in oil or PFC.
The responses to all the dried preparations was lower than the fresh vaccine
control (not shown) indicating a significant loss of immunogenicity on spray
drying.
The antigenicity of the toxoid, as measured by capture ELISA, was unaltered by
the
drying process. This suggested that more work is required to perfect the
preservation
of the aluminum hydroxide adjuvant on drying.
The response to STASIS vaccine density-matched with calcium phosphate
(group 3) is essentially the same as the control vaccine reconstituted in
aqueous
buffer (group 1) and the powder in oil vaccine (group 2) while control animals
injected with the non-aqueous vehicles only (groups 4 & 5) showed no response.
Description of Preferred Embodiments
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EXAMPLE 1:
Spray-dried particles in PFCs.
Particles were produced by spray drying from aqueous solution using a
Labplant model SD 1 spray dryer using sugars and other excipients. Typical
formulations were:
A. mannitol 15% w/v
calcium lactate 15% w/v
in water
B. trehalose 15% w/v
calcium phosphate 15% w/v
in water
ftte particles were produced using a two-fluid nozzle with a liquid orifice of
0.5 mm internal diameter. A half-maximum nozzle airflow was found optimal and
the drying chamber operated at an inlet temperature of 135 C and an outlet
temperature of 70-75 C. The particles were collected in a glass cyclone and
subjected
to secondary drying in a vacuum oven using a temperature ramp to 80 C over 4
hours.
On cooling they were suspended in PFC using ultrasound. Either a 30 second
burst
of ultrasonic energy from a titanium probe in an MSE MK 2 ultrasonic cabinet
operating at about 75% power or immersion in a Decon FS200 Frequency sweep
Ultrasonic bath for up to 10 minutes was found to be sufficient.
The resulting suspension was monociisperse and consisted of spherical glass
particles ranging in size from about 0.5 to 30 j.t, with a mean of about 10
as judged
microscopically. The mannitol / calcium lactate particles rose to the top of
the PFC
layer over several minutes but could readily be resuspended with gentle
shaking. The
trehalose / calcium phosphate particles were almost density matched with the
PFC
and formed a stable suspension.
Spray dried powders of sugar glass particles were suspended in
perfluorohexane, perfluorodecalin and perfluorophenanthrene at 1, 10, 20 and
40%
w/v. They were found to give monodisperse suspensions with little tendency to
aggregate. The addition of 0.1% perfluorodecanoic acid to the PFC inhibited
any
CA 02689856 2010-01-13
slight tendency to aggregate on surfaces. These suspensions were found to pass
easily through a 25 g needle by aspiration or ejection.
EXAMPLE 2:
Stability of suspension of glass stabilized enzyme in PFC
5 Alkaline
phosphatase (Sigma Aldrich Ltd.) was spray dried in a Labplant machine as
above. The formulation contained mannitol 33.3% w/w, calcium phosphate 33.3%
w/w and degraded gelatin (Byco C, Croda colloids Ltd.) 33.3%. The dried enzyme
was stored at 55 C either as the dry powder or as a suspension in
perfluorodecalin.
The enzyme formulated in these microspheres consisting of a Mannitol-based
glass
10 suspended in
perfluorodecalin show retention of close to 100% of enzyme activity for
more than 30 days at 55 C (Fig 1).
EXAMPLE 3:
Efficacy in vivo
Pre clinical trials of a similar formulation containing a clinical tetanus
toxoid
15 vaccine
(kindly supplied by Medeva plc) were undertaken in collaboration with the
National Institute of Biological standards and Control (an approved laboratory
of the
World Health Organization). The results from this trial showed that the stable
STASIS preparation was completely equivalent to a water-based liquid vaccine
in its
ability to immunize guinea pigs to develop a protective serum antibody
response
(Figure 2). This confirmed that the suspension in PFC constituted a ready-to-
inject
formulation with the same bioavailability in vivo as a conventional water
based liquid
formulation.
EXAMPLE 4:
Spray-freeze-dried particles
Particles were also made by spraying liquid droplets into liquid nitrogen and
then vacuum-drying the frozen powder. These particles were less dense than the
spray dried powders and formed pastes in PFCs at concentrations higher than
20%
w/v. At lower concentrations they formed monodisperse suspension after
sonication.
Typical formulations used were
substance final concentration w/w
A. Trehalose 100%
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B. Trehalose 50%
Calcium phosphate 49.5%
Aluminum hydroxide 0.5%
EXAMPLE 5:
Milled hydrophobic particles
The hydrophobic sugar derivatives sucrose octaacetate and trehalose
octaacetate readily form glasses when either quenched from the melt or dried
rapidly
from solution of chloroform or dichloromethane. Their use has been described
as
controlled release matrices for drug delivery (Roser et al "Solid delivery
systems for
controlled release of molecules incorporated therein and methods of making
same"
PCT Pub No WO 96/03978 1994)
A trehalose octaacetate powder was made by melting in a muffle furnace and
quenching the melt on a stainless steel plate. The resultant glass disks were
ground
in a pestle and mortar and then in a high-speed homogenizer to produce a fine
powder. This was
suspended in perfluorohexane, perfluorodecalin and
perfluorophenanthrene at 1 and 10% w/v. They were found to give well dispersed
suspensions. These suspensions were found to pass easily through a 23g needle.
EXAMPLE 6:
Reconstitution in aqueous environments
Because of the nature of the soluble sugar glass particles and the properties
of the PFCs it was anticipated that actives in these suspensions would release
rapidly
in the body. In order to demonstrate complete release of an included active
substance, particles were formulated to contain:
Trehalose 20% w/v
Calcium lactate 20% w/v
Lysine 0.5% w/v
Mordant Blue 9 dye 1% w/v
The formulation was spray dried as above and added to
perfluorophenanthrene and perfluorodecalin to produce 20% w/v dark blue,
opaque
suspensions. Upon addition of water to an equal volume of the suspensions and
shaking, it was found that virtually all of the blue dye was released into the
water
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phase which formed a clear blue layer floating on the nearly colorless PFC
with a
clean sharp interface between them.
EXAMPLE 7:
Non reactivity between particles in suspension
Because the individual microspheres in a PFC suspension are physically
isolated from all other particles, potentially reactive substances can be
present
together in the same suspension in separate particles without any danger of
them
interacting. When the sugar glass is dissolved and the molecules can come
together,
the reaction occurs.
In order to demonstrate this, a suspension was made to contain two types of
particles, one (a) with the enzyme alkaline phosphatase and the other (b) with
its
colorless substrate, paranitrophenyl phosphate
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Formulations were:
a) Trehalose 10% w/v
Sodium sulphate 10% w/v
Alkaline phosphatase 20 Um]
In 5 mM Tris / HC1 buffer pH 7.6
b) Trehalose 10% w/v
Sodium sulphate 10% w/v
Parani trophenyl phosphate 0.44% w/v
In 100 mM Glycine buffer pH 10.2 containing 1 rnM each of Zn++ and Mg'
Chloride
A suspension of the powders in perfluorodecalin containing 10% w/v of
powder "a" and 10% w/v of powder "b" was found not to develop any color
reaction
but to remain as a white suspension for 3 weeks at 37 C.
Upon the addition of water and shaking, the powders dissolved in the
overlying aqueous phase. The enzyme reaction took place in a mater of minutes,
producing an intense yellow color of p-nitrophenol, both in the freshly
prepared
sample and in that which had been kept at 37 C for 3 weeks.
EXAMPLE 8:
Product release in model "tissue space"
In order to illustrate the possible behavior of PFC suspensions when injected
in vivo, a model, transparent, hydrated tissue space was prepared by casting
0.2%
agarose gels in polystyrene bijoux bottles. 0.1m1 of the perfluorodecalin
suspension
from example 5 was injected through a 25g needle into the agarose gel. This
produced a flattened white sphere of the suspension. Over the next 5-10
minutes the
white color cleared from the bottom of the sphere upwards leaving a clear
sphere of
PFC behind. As the enzyme and substrate were released by the dissolution of
the
glass particles, they reacted together producing a yellow color of p-ni
trophenol, which
then diffused throughout the agarose over the next 1 hr.
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EXAMPLE 9:
Density matching.
Sugar glass particles (i.e. trehalose) obtained by either of the conventional
drying methods show typical densities around 1.5 g/cm3. The Perfluorocarbons
we
tested typically have densities ranging from 1.68 to 2.03 g/cm3 (Table I). For
this
reason when formulated into a suspension, sugar glass particles tend to float
on the
PFC layer, leading to a preparation in which the active is not homogeneously
distributed. Powders may however be modified in order to produce a stable
suspension in PFC in which they have neutral buoyancy and neither settle nor
float.
This may be achieved through the addition of high-density materials prior to
particle
formation. These may be water soluble or insoluble.
Nor. water-soluble materials
Tricalcium orthophosphate has a density of 3.14 gicm3, is approved as an
adjuvant for vaccines and is practically insoluble in water. Powders made to
contain
around 50% calcium phosphate show an increased density around 2 g/cm3 and at
20%
solids form stable suspensions in perfluorophenanthrene.
Examples of powders which at 20% solids in PFCs form stable suspensions
include:
1 in perfluorodecalin
substance final concentration w/w
A. Trehalose 50%
Calcium phosphate 50%
B. Trehalose 47.5%
Calcium lactate 10.0%
Calcium phosphate 42.5%
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2 in perfluorophenanthrene:
substance final concentration w/w
Mannitol 18.2%
Inositol 18.2%
5 Calcium lactate 18.2%
Calcium phosphate 45.4%
Other density increasing non water-soluble materials, which have been used,
include barium sulphate and titanium dioxide. Any non-toxic and insoluble
material
with the appropriate density can be used.
10 Water soluble materials
Soluble salts such as sodium sulphate with a density of 2.7 g/cm3may also be
used as a density-increasing agent. The following powder formed stable
suspensions
in perfluorodecalin:
substance final concentration w/w
15 Trehalose 50%
Sodium sulphate 50%
Other non-toxic high-density water soluble materials can also be used. These
formulations have been found to cause discomfort after subcutaneous injection
in
guinea pigs, possibly because of the rapid dissolution of high concentrations
of ionic
20 salt.
EXAMPLE 10:
Effect of density matching on actives in suspensions
Certain vaccines are formulated adsorbed on to insoluble gels or particles
which act as adjuvants. Aluminum hydroxide and calcium phosphate are
extensively
used for this purpose. These insoluble adjuvants may themselves be used to
increase
the density of the particles to be suspended. hi this case the high-density
material is
not completely inert but in fact adsorbs the active macromolecule from
solution. It
is necessary to demonstrate that this adsorption does not denature the active.
To test
this, alkaline phosphatase was used as a model active/vaccine.
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The following solution was made
Adjuvant grade calcium phosphate 10% w/v (Superphos Kemi a/s)
Trehalose 10% w/v
ZnC12 1 mM
MgC12 1 rtiM
Alkaline phosphatase 20 U/ml
In 5mM Tris HCI buffer pH 7.6
The solution was then well mixed for 10 minutes at 37 C to allow the alkaline
phosphatase to be adsorbed by the calcium phosphate. This change in absorption
per
minute was measured by centrifuging the calcium phosphate, sampling the
supernatant and measuring its enzyme kinetics using p-nitrophenyl phosphate as
substrate and a wavelength of 405nm. The solution was spray-dried to produce a
fine
powder. Any desorption of the enzyme after rehydrati on of the powder was
measured
in the supernatant as above. The powder was suspended at 20% w/v in
perfluorophenanthrene and found to produce a stable suspension.
Sample tested dAls0 /min
.(405n m).
Original solution (25 1) 0.409
Supernatant from above (25 p.1) 0.034
Rehydrated powder (25_ 1 of a 20%w/v in water) 0.425
Supematant from above (25 pl) 0.004
20%w/v powder in perfluorodecalin (25 I) 0.430
The experiment demonstrates:
The density of the particles may be matched to that of the PFC vehicle by the
inclusion of the adjuvant calcium phosphate.
No significant desorption or loss of enzyme activity takes place during the
formulation process.
EXAMPLE 11:
A STASIS preparation of the mannitol base glass as in example 1 was
suspended in perfluorodecalin and loaded into a surgically clean, pump-action,
polypropylene atomizer which is normally used clinically to deliver
oxymetazoline
nasal decongestant (Sudafed, Warner Lambert). Two sprays of the suspension
were
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delivered into each nostril of a human volunteer who were asked to comment on
the
degree of discomfort experienced. The volunteer reported no discomfort at all.
There was no observable side effects of the administration.
