Note: Descriptions are shown in the official language in which they were submitted.
WO96/09814 PCT/GB95l02279
_ 1 2 1 g 9 ~ 5 4
SPRAY-DRIE~ MICROPARTICLES AS THERAPEUTIC VEHICLES
Field of the Invention
This invention relates to spray-dried microparticles
and their use as therapeutic vehicles. In particular, the
invention relates to means for delivery of diagnostic and
therapeutic agents and biotechnology products, including
therapeutics based upon rDNA technology.
Backqround of the Invention
The most commonly used route of administration of
therapeutic agents, oral or gastrointestinal, is largely
inapplicable to peptides and proteins derived from the rDNA
industry. The susceptibility of normally blood-borne
peptides and proteins to the acidic/proteolytic environment
of the gut, largely precludes this route for
administration. The logical means of administration is
intravenous, but this presents problems of poor patient
compliance during chronic administration and very often
rapid first-pass clearance by the liver, resulting in short
iv lifetimes.
Recently, the potential for delivery by mucosal
transfer has been explored. Whilst nasal delivery has been
extensively explored, the potential delivery of peptides
via the pulmonary airways is largely unexplored.
Alveolar cells, in their own right, provide an
effective barrier. However, even passage of material to
the alveolar region represents a significant impediment to
this method of administration. There is an optimal size of
particle which will access the lowest regions of the
pulmonary airways, i.e. an aerodynamic diameter of <5 ~m.
Particles above this size will be caught by impaction in
the upper airways, such that in standard commercial
suspension preparations, only 10-30% of particles, from
what are normally polydispersed suspensions, reach the
lowest airways.
Current methods of aerosolising drugs for inhalation
include nebulisation, metered dose inhalers and dry powder
systems. Nebulisation of aqueous solutions requires large
WO 96/09814 P~ b5slo2279
02 1 99 954
volumes of drugs and involves the use of bulky and non-
portable devices.
The most common method of administration to the lung
is by the use of volatile propellant-based devices,
commonly termed metered dose inhalers. The basic design is
a solution of propellant, commonly CFC 11, 12 or 114,
containing either dissolved drug or a suspension of the
drug in a pressurised canister. Dosing is achieved by
depressing an actuator which releases a propellant aerosol
of drug suspension or solution which is carried on the
airways. During its passage to the lung, the propellant
evaporates to yield microscopic precipitates from solution
or free particles from suspension. The dosing is fairly
reproducible and cheap, but there is growing environmental
pressure to reduce the use of CFCs. Furthermore, the use
of CFC solvents remains largely incompatible with many of
the modern biotechnology drugs, because of their
susceptibility to denaturation and low stability.
Concurrently, there is a move toward dry powder
devices which consist of dry powders of drugs usually
admixed with an excipient, such as lactose or glucose,
which facilitates the aerosolisation and dispersion of the
drug particles. The energy for disaggregation is often
supplied by the breath or inspiration of air through the
device.
Drugs are currently micronised, to reduce particle
size. This approach is not applicable for biotechnology
products. In general, biotechnology products are available
in low quantity and, furthermore, are susceptible to the
methods currently employed to dry and micronise prior to
mixing with excipient. Further, it is particularly
difficult to provide blends of drug and excipient which are
sufficiently free-flowing that they flow and dose
reproducibly in the modern multiple dose inhalers such as
the Turbohaler (Astra) and Diskhaler (Glaxo). Studies have
revealed that, contrary to expectation, spray-dried
(spherical) salbutamol microparticles showed greater forces
WO96/098l4 P~-l/~b55~27~
-
3 0 2 1 9 ~ 9 5 4
of cohesion and adhesion than similarly-sized particles of
micronised drug. Electron micrographs of the spray-dried
material revealed the particles to possess pitted, rough
surfaces.
Haghpanah et al reported, at the 1994 British
Pharmaceutical Conference, that albumin microparticles
incorporating salbutamol, had been produced by spray-drying
and were of a suitable size for respiratory drug delivery,
i.e. 1-5 ~m. The aim was to encapsulate salbutamol, for
slow release. It does not appear that the product is of
substantially uniformly spherical or smooth microparticles
that have satisfactory flow properties, for multi-dose dry
powder inhalers.
Diagnostic agents comprising hollow microcapsules have
been used to enhance ultrasound imaging. For example, EP-
A-458745 (Sintetica) discloses a process of preparing air-
or gas-filled microballoons by interfacial polymerisation
of synthetic polymers such as polylactides and
polyglycolides. W0-A-9112823 (Delta) discloses a similar
process using albumin. Wheatley et al (1990) Biomaterials
11:713-717, disclose ionotropic gelation of alginate to
form microbubbles of over 30 ~m diameter. W0-A-9109629
discloses liposomes for use as ultrasound contrast agents.
Przyborowski et al, Eur. J. Nucl. Med. 7:71-72 (1982),
disclose the preparation of human serum albumin (HSA)
microspheres by spray-drying, for radiolabelling, and their
subsequent use in scintigraphic imaging of the lung. The
microspheres were not said to be hollow and, in our
repetition of the work, predominantly poorly formed solid
microspheres are produced. Unless the particles are
hollow, they are unsuitable for echocardiography.
Furthermore, the microspheres were prepared in a one-step
process which we have found to be unsuitable for preparing
- microcapsules suitable for echocardiography; it was
necessary in the prior process to remove undenatured
albumin from the microspheres, and a wide size range of
WO96/09814 0 2 1 9 9 9 ~ b~ 279
microspheres was apparently obtained, as a further sieving
step was necessary.
Przyborowski et al refer to two earlier disclosures of
methods of obtaining albumin particles for lung
scintigraphy. Aldrich & Johnston (1974) Int. J. Appl. Rad.
Isot. 25:15-18, disclose the use of a spinning disc to
generate 3-70 ~m diameter particles which are then
denatured in hot oil. The oil is removed and the particles
labelled with radioisotopes. Raju et al (1978)
Isotopenpraxis 14(2):57-61, used the same spinning disc
technique but denatured the albumin by simply heating the
particles. In neither case were hollow microcapsules
mentioned, and the particles prepared were not suitable for
echocardiography.
EP-A-0606486 (Teijin) describes the production of
powders in which an active agent is incorporated into small
particles, with carriers comprised of cellulose or
cellulose derivatives. The intention is to prevent drug
particles from adhering to the gelatin capsules used in a
unit dose dry powder inhaler. Page 12 of this publication
refers to the spray-drying of "medicament and base", to
obtain particles of which 80% or more were 0.5-10 ~m in
size. No directions are given as to what conditions should
be used, in order to obtain such a product.
EP-A-0611567 (Teijin) is more specifically concerned
with the production of powders for inhalation, by spray-
drying. The carrier is a cellulose, chosen for its
resistance to humidity. The conditions that are given in
Example 1 (ethanol as solvent, 2-5% w/v solute) mean that
there is no control of surface morphology, and Example 4
reports a poor lower airway respirable fraction (12%),
indicating poor dispersion properties. Spherical particles
are apparently obtained at high drug content, indicating
that particle morphology is governed by the respective drug
and carrier contents.
Conte et al (1994) Eur. J. Pharm. Biopharm. 40(4):203-
208, disclose spray-drying from aqueous solution, with a
WO96/09814 2 1 9 9 9 5P~ /~b5~ >79
maximum solute concentration of 1.5%. High drug content is
required, in order to obtain the most nearly spherical
particles. This entails shrunken and wrinkled particle
morphology. Further, after suspension in butanol, to
facilitate Coulter analysis, sonication is apparently
necessary, implying that the particles are not fully dry.
It is an object behind the present invention to
provide a therapeutic delivery vehicle and a composition
that are better adapted than products of the prior art, for
delivery to the alveoli in particular.
SummarY of the Invention
According to the present invention, it has
surprisingly been found that, in microparticles (and also
microcapsules and microspheres) that are also suitable as
an intermediate product, i.e. before fixing, in the
production of air-containing microcapsules for diagnostic
imaging, e.g. as disclosed in WO-A-9218164 as "intermediate
microcapsules", the wall-forming material is substantially
unaffected by spray-drying. Thus, highly uniform
microparticles, microspheres or microcapsules of heat-
sen~itive materials such as enzymes, peptides and proteins,
e.g. HSA, and other polymers, may be prepared and
formulated as dry powders, for therapeutic or diagnostic
use.
By contrast to the prior art, it has also now been
found that effective, soluble carriers for therapeutic and
diagnostic agents can be prepared, by spray-drying, and
which are free-flowing, smooth, spherical microparticles of
water-soluble material, e.g. human serum albumin (HSA),
having a mass median particle size of 1 to 10 ~m. More
generally, a process for preparing microcapsules of the
invention comprises atomising a solution (or dispersion) of
a wall-forming material. A therapeutic or diagnostic agent
may be atomised therewith, or coupled to the microcapsules
thus produced. Alternatively, the material may be an
active agent itself. In particular, it has been found
that, under the conditions stated herein, and more
WO96/09814 PCT/GB95/02279
6 2 1 9 9 9 5 4
generally described by Sutton et al (1992), e.g. using an
appropriate combination of higher solute concentrations and
higher air:liquid flow ratios than Haghpanah et al, and
shell-forming enhancers, remarkably smooth spherical
microparticles of various materials may be produced. The
spherical nature of the microparticles can be established
by means other than mere maximum size analysis, i.e. the
laser light diffraction technique described by Haghpanah et
al. Moreover, the particle size and size distribution of
the product can be controlled within a tighter range, and
with greater reproducibility. For example, by Coulter
analysis, 98% of the particles can be smaller than 6 ~m on
a number basis, within an interquartile range of 2 ~m, and
with less than 0.5 ~m mean size variation between batches.
Furthermore, when tested in a dry powder inhaler under
development, reproducible dosing was achieved, and
subsequent aerosolisation under normal flow conditions (30
l/min) resulted in excellent separation of microparticles
from excipient.
Unfixed capsules of this invention, composed of non-
denatured HSA or other spray-dryable material, possess
highly smooth surfaces and may be processed with relatively
low levels of excipients to produce free-flowing powders
ideal for dry powder inhalers. Using this approach, it is
possible to produce heterogeneous microcapsules which are
comprised of a suspending excipients and active principle.
This has the advantage of yielding free-flowing powder of
active principles which may be further processed to give
powders that dose and aerosolise with excellent
reproducibility and accuracy.
In addition, the process of spray-drying, in its
current form, gives rise to relatively little denaturation
and conversion to polymers in the production of the free-
flowing powder. In all cases, the size of the microcapsule
suspension can be such that 90% of the mass lies within the
desired size, e.g. the respirable region of 1-5 ~m.
WO96/09814 PCTIGB95/02279
7 0 2 1 9 ~ ~ 5 4
In essence therefore we have defined how to produce
microparticles which are: predominantly 1-5 ~m in size;
smooth and spherical; gas-containing; and composed of
undamaged protein molecules and which may be stored and
shipped prior to other processing steps. In preparing
intermediate microcapsules for ultrasound imaging, we have
defined those characteristics of a process and the
resulting powder which are essential for the production of
superior powders for dry powder inhalers (DPI's). We find
that many of the assays which have been developed for the
echocontrast agent are suitable for defining those
parameters of the particles which are advantageous for DPI
powders, namely; echogenicity and pressure resistance of
cross-linked particles defining perfectly formed
microparticles; microscopic evaluation in DPX or solvents,
defining sphericity and gas-containing properties of
soluble intermediate capsules; size and size distribution
analysis and also the monomeric protein assay to define the
final level of fixation of the product.
Especially for use in therapy, considerable care is
necessary in order to control particle size and size
distribution. We have chosen a biocompatible polymer which
when cross-linked remains innocuous and also learned how to
reproducibly cross-link this molecule. In order to achieve
controlled cross-linking, we have divorced the processes of
microparticle formation and cross-linking which other
emulsion and solvent evaporation process do not. This
means that the initial step of the process does not damage
the wall-forming material. We have defined the particular
parameters which are important for complete particle
formation and further defined more advantageous conditions
which yield more intact particles. In choosing HSA as a
particularly favourable polymer we have also chosen a
- potential carrier molecule which may: protect labile
molecules; enhance uptake of peptides across the lung;
bind low molecular weight drug through natural binding
affinities; and be covalently modified to carry drugs
WO96/09814 2 1 9 9 9 ~C4T/GB95/02279
across cellular barriers to the systemic circulation and
beyond.
When researchers have used spray-drying to produce
microparticles of small dimensions, they have tended to use
volatile solvents, which encourages rapid droplet
shrinkage. Alternatively, researchers have used feedstocks
with low solute content in order to keep the solution
viscosity low, to enhance smaller droplet production. In
both cases, when the microparticles are produced, the
process has little impact on the final morphology; rather
this is dictated by the components used to form the
particles. We have taken the extensive learning of how to
produce controlled sized particles from HSA and applied
this to many other materials including active drugs. We
are able to use relatively high solute contents, e.g. l0-
30% w/v as opposed to 0.5-2%, to produce microparticles
comprising low molecular weight active with lactose; active
alone: peptides with HSA and modified polymeric carriers
with active. We now find that it is the process which
dictates the final particle morphology rather than the
composition of the solutes. Further, we are able to use
combinations of aqueous and water-miscible solvents to
enhance particle morphology. Thus we have a process~
driven methodology which allows beneficial production of
smooth, spherical controlled sized particles suitable for
pulmonary delivery.
It has been found that the process of the invention
can be controlled in order to obtain microspheres with
desired characteristics. Thus, the pressure at which the
protein solution is supplied to the spray nozzle may be
varied, for example from l.0-l0.0 x 105 Pa, preferably 2-8
x l05 Pa, and most preferably about 7.5 x l0 Pa. Other
parameters may be varied as disclosed below. In this way,
novel microspheres may be obtained.
A further aspect of the invention provides hollow
microspheres in which more than 30%, preferably more than
40%, 50%, or 60%, of the microspheres have a diameter
WO96/098l4 PCT/GB95/02279
0 2 1 9 ~ 9 5 4
within a 2 ~m range and at least 90%, preferably at least
95% or 99%, have a diameter within the range 1.0-8.0 ~m.
The interquartile range may be 2 ~m, with a median
diameter of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5 ~m.
Thus, at least 30%, 40~, 50~ or 60% of the
microspheres may have diameters within the range 1.5-3.5
~m, 2.0-4.0 ~m, 3.0-5.0 ~m, 4.0-6.0 ~m, 5.0-7.0 ~m or 6.0-
8.0 ~m. Preferably a said percentage of the microspheres
have diameters within a 1.0 ~m range, such as 1.5-2.5 ~m,
2.0-3.0 ~m, 3.0-4.0 ~m, 4.0-5.0 ~m, 5.0-6.0 ~m, 6.0-7.0 ~m
or 7.0-8.0 ~m.
A further aspect of the invention provides hollow
microspheres with proteinaceous walls in which at least
90%, preferably at least 95% or 99%, of the microspheres
have a diameter in the range 1.0-8.0 ~m; at least 90%,
preferably at least 95% or 99%, of the microspheres have a
wall thickness of 40-500 nm, preferably 100-500 nm.
Descri~tion of the Invention
The wall-forming material and process conditions
should be so chosen that the product is sufficiently non-
toxic and non-immunogenic in the conditions of use, which
will clearly depend on the dose administered and duration
of treatment. The wall-forming material may be a starch
derivative, a synthetic polymer such as tert-butyloxy-
carbonylmethyl polyglutamate (US-A-4888398) or a
polysaccharide such as polydextrose.
Generally, the wall-forming material can be selected
from most hydrophilic, biodegradable physiologically
compatible polymers, as described in more detail in WO-A-
9218164.
Preferably, the wall-forming material is
proteinaceous. For example, it may be collagen, gelatin or
(serum) albumin, in each case preferably of human origin
(i.e. derived from humans or corresponding in structure to
the human protein). Most preferably, it is human serum
albumin (HSA) derived from blood donations or, ideally,
from the fermentation of microorganisms (including cell
WO 96/09814 ~ ,;b55/02279
0 2 1 9 9 9 5 4
lines) which have been transformed or transfected to
express HSA. Further detail is given in W0-A-9218164.
The protein solution or dispersion is preferably 0.1
to 50% w/v, more preferably about 5.0-25.0% protein,
particularly when the protein is albumin. About 20% is
optimal. Mixtures of wall-forming materials may be used,
in which case the percentages in the last two sentences
refer to the total content of wall-forming material.
The preparation to be sprayed may contain substances
other than the wall-forming material and solvent or carrier
liquid. Again, reference may be made to W0-A-9218164.
The protein solution or dispersion (preferably
solution), referred to hereinafter as the "protein
preparation", is atomised and spray-dried by any suitable
technique which results in discrete microspheres or
microcapsules of 1 to 10 ~m diameter. These figures refer
to at least 90% of the population of microcapsules, the
diameter being measured with a Coulter Master Sizer TI.
The term "microcapsules" means hollow particles enclosing
a space, which space is filled with a gas or vapour but not
with any solid materials. Honeycombed particles resembling
the confectionery sold in the UK as Maltesers~ are not
formed.
The atomising comprising forming an aerosol of the
protein preparation by, for example, forcing the
preparation through at least one orifice under pressure
into, or by using a centrifugal atomiser in a chamber of
warm air or other inert gas. The chamber should be big
enough for the largest ejected drops not to strike the
walls before drying. The gas or vapour in the chamber is
clean (i.e. prefera~ly sterile and pyrogen-free) and non-
toxic when administered into the bloodstream in the amounts
concomitant with administration of the microcapsules in
use. The rate of evaporation of the liquid from the
protein preparation should be sufficiently high to form
hollow microcapsules but not so high as to burst the
microcapsules. The rate of evaporation may be controlled
WO96/09814 PCT/GB95/02279
11 0 2 1 9 ~ 9 5 4
by varying the gas flow rate, concentration of protein in
the protein preparation, nature of liquid carrier, feed
rate of the solution and, more importantly, the temperature
of the gas encountered by the aerosol. With an albumin
concentration of 15-25% in water, an inlet gas temperature
of at least about 100C, preferably at least 110C, is
generally sufficient to ensure hollowness and the
temperature may be as high as 250C without the capsules
bursting. About 180-240C, preferably about 210-230C and
most preferably about 220C, is optimal, at least for
albumin. Since the temperature of the gas encountered by
the aerosol will depend also on the rate at which the
aerosol is delivered and on the liquid content of the
protein preparation, the outlet temperature may be
monitored to ensure an adequate temperature in the chamber.
An outlet temperature of 40-150C has been found to be
suitable. Controlling the flow rate has been found to be
useful in controlling the other parameters such as the
number of intact hollow particles.
The microcapsules comprise typically 96-98% monomeric
HSA.
More particularly, microparticles of the invention
preferably have a maximum interquartile range of 3 ~m, more
preferably 2 ~m, and most preferably 1.5 ~m, with respect
to their mass median particle size. The mass median
particle diameter is determined by Coulter counter with a
conversion to a volume-size distribution. This is achieved
by spray-drying in which there is a combination of low feed
stock flow rate with high levels of atomisation and drying
air. The effect is to produce microcapsules of very
defined size and tight size distribution.
Several workers have designed equations to define the
mean droplet size of pneumatic nozzles; a simple version of
- the various parameters which affect mean droplet size is as
follows:
D = A/(V d) + B.(Majr/M,jq)
WO96/09814 PCT/GB95102279
12 021 99 ~54
Where D = Mean droplet size
A = Constant related to nozzle design
B = Constant related to liquid viscosity
V = Relative air velocity between liquid and
nozzle
d = Air density
Majr and M~jq = Mass of air and liquid flow
a and b = Constants related to nozzle design
Clearly, for any given nozzle design, the droplet size
is most affected by the relative velocity at the nozzle and
concurrently the mass ratio of air to liquid. For most
common drying use, the air to liquid ratio is in the range
of 0.1-10 and at these ratios it appears that the average
droplet size is 15-20 ~m. For the production of
microparticles in the size range described herein we use an
air to liquid ratio ranging from 20-1000:1. The effect is
to produce particles at the high ratios which are
exceedingly small by comparative standards, with very
narrow size distributions. For microparticles, produced at
the lower ratios of air to liquid, slightly larger
particles are produced, but they still nevertheless have
tight size distributions which are superior to
microparticles produced by emulsion techniques.
The amount of added active principle is not critical;
the microparticles may comprise at least 50, more
preferably 70 or 80, and most preferably 90, % by weight
HSA or other carrier material. For use in an inhaler
device, the microparticles may be formulated with a
conventional excipient such as lactose or glucose.
The microparticles may comprise therapeutic agent and
carrier, or a compound which alone is therapeutically-
active. The amount of the active principle will be chosen
having regard to its nature and activity, to the mode of
administration and other factors known to those skilled in
the art. By way of example only, the number of particles
administered may be such as to deliver 100 mg/day ~-1 anti-
W096/09814 PCTt~b951~727~
_ 13 2 1 9 9 ~ 5 4
trypsin, or 0.1 mg/day of an active material such asbeclomethasone.
The active principle may be, for example, a diagnostic
substance or a classical pharmaceutical entity which may or
may not bind, covalently or otherwise, to the carrier
material. The therapeutic agent may be a proteinaceous
material such as insulin, parathyroid hormone, calcitonin
or similar bioactive peptide, albuterol, salicylate,
naproxen, augmentin or a cytotoxic agent. For experimental
purposes, a marker such as lysine-fluorescein may be
included.
Microparticles of the invention may comprise an
antagonist or receptor-binding component in addition to the
therapeutic or diagnostic agent. For example, a sugar or
lS other molecule may be included in the molecular vehicle,
with a view to directing administration of the vehicle-
bound drug to a given receptor at or beyond the alveoli.
HSA is used herein as an illustrative example of
water-soluble carrier materials for use in the invention.
Other materials that can be used include simple and complex
carbohydrates, simple or complex amino- or polyamino-acids,
fatty acid or fatty acid esters, or natural or recombinant
human proteins or fragments or short forms thereof.
The invention allows for the nature of the dry
microcapsules to be manipulated, in order to optimise the
flow or vehicle properties, by changing and reducing the
forces of cohesion and adhesion within the microcapsule
preparation. For example, if so required, the
microcapsules may be made predominantly positive or
negative by the use of highly-charged monomeric or
polymeric materials, e.g. lysine or poly-lysine and
glutamate or poly-glutamate in systems without HSA or
heterogeneous systems including HSA and active principles.
A further embodiment of the invention is the co-spray-
drying of the active principle with HSA in order tofacilitate stabilisation of the active principle during
formulation, packing and, most importantly, during
W0 96/09814 ~ 5~b9~ >79
14
residence on the alveolar lining. In this environment,
there can be intense proteolytic activity. Whilst protease
inhibitors can be used to protect peptide drugs, there may
well be contra-indications to this approach. By using HSA,
both as excipient and vehicle, it can offer a large excess
of alternative substrate on which the locally-active
proteases may act. A further advantage is that, since HSA
has been shown to cross the alveolar barrier, by receptor-
or non-receptor-mediated transcytotic mechanisms, it may be
used as a vehicle to facilitate the passage of an active
principle across the epithelial lining.
In a further embodiment, active principle may be
covalently linked to HSA via cleavable linkages prior to
spray-drying. This embodiment represents a method of
carrying active principles all the way from device to
bloodstream, and possibly to targets within the body. The
formation of particles with optimal aerodynamic size means
that the "physical" vehicle delivers the active principle
to the site of absorption. Once deposited upon the
alveoli, the "molecular" vehicle then protects and
facilitates passage into the bloodstream and, once in the
bloodstream, can further enhance circulatory half-life and
even direct the active principle to certain sites within
the body on the basis of receptor-mediated events.
A suitable linker technology is described in WO-A-
9317713 (Rijksuniversiteit Groningen). Esterase-sensitive
polyhydroxy acid linkers are described. Such technology,
used in the derivatisation of HSA prior to spray-drying,
enables the production of a covalent carrier system for
delivery of drugs to the systemic vasculature. This
utilises the potential of HSA to cross the alveoli to carry
drugs over a prolonged period whilst protecting potentially
unstable entities.
Although the active principle used in this invention
may be imbibed into or otherwise associated with the
microparticles after their formation, it is preferably
formulated with the HSA. The microparticles may be at
WO96/09814 PCT/GB95/02279
2 1 9 ~ 9 5 4
least partly coated with a hydrophobic or water-insoluble
material such as a fatty acid, in order to delay their rate
of dissolution and to protect against hydroscopic growth.
The following Examples illustrate the invention. The
spray dryer used in the Examples, available from A/S Niro
Atomizer, Soeborg, Denmark, under the trade name "Mobile
Minor", is described in detail in WO-A-9218164.
Exam~le 1
A 20% solution of sterile, pyrogen-free HSA in
pyrogen-free water (suitable for injection) was pumped to
the nozzle of a two-fluid nozzle a~tomizer mounted in the
commercial spray-drying unit described above. The
peristaltic pump speed was maintained at a rate of
approximately 10 ml/minute such that with an inlet air
temperature of 220C the outlet air temperature was
maintained at 95C.
Compressed air was supplied to the two fluid
atomising nozzle at 2.0-6.0 Bar (2.0-6.0 x 10 Pa). In
this range microcapsules with a mean size of 4.25-6.2 ~m
are obtained.
Typically an increase in mean particle size (by
reduced atomisation pressure) led to an increase in the
amount of microcapsules over 10 ~m in size (see Table 1).
Table 1
Effects of Atomisation Pressure on Frequency of
Microcapsules Over 10 ~m in Diameter
Atomisation Pressure% Frequency over 10 mm
(x105 Pa)
6.0 0.8
5.0 3.3
3.5 6.6
2.5 8.6
2.0 13.1
Under the conditions described above, i.e. the first
step of Example 1 of WO-A-9218164, with a nozzle pressure
WO96/09814 PCT/~b~S~279
16 ~ ~ 1 9 ~ ~4
of 7.5 bar, microparticles were produced with a particle
size of 4.7 ~m. These soluble microparticles were smooth
and spherical with less than 1~ of the particles over a
particle size of 6 ~m. The microparticles were dissolved in
aqueous medium and the molecular weight of the HSA
determined by gel filtration chromatography. The resultant
chromatograms for the HSA before and after spray-drying HSA
are essentially the same. Further analysis of the HSA
before and after spray-drying by means of tryptic peptide
mapping with HPLC revealed that there were no observable
differences in the peptides liberated. Both analyses show
that, under the conditions of spray-drying described to
produce microparticles of 4.7 ~m, little or no structural
damage is done to the protein.
ExamPle 2
Alpha-1 antitrypsin derived from human serum was
spray-dried under conditions similar to Example 1 with an
inlet temperature of 150C and an outlet temperature of
80C. In all other respects the conditions for drying were
the same as Example 1. The soluble microparticles produced
had a mean size of 4.5 ~m. The microparticles were
dissolved in aqueous medium and analysed for retention of
protein structure and normal trypsin inhibitory activity,
then compared to the original freeze dried starting
material. Analysis by gel permeation and reverse phase
chromatography and capillary electrophoresis, revealed that
there were no significant structural changes after spray-
drying. Analysis of the inhibitory activity (Table 2)
showed that within the experimental error, full retention0 of inhibitory activity had been achieved.
Table 2
Run Number Percentage of Activity
Retained
1 84
2 222
3 148
WO96/09814 PCT/~b~S~ 79
~ 17 0 2 1 9 9 9 5 4
Exam~le 3
Using the general method of Example 1, microcapsules
composed of alcohol dehydrogenase (ADH) and lactose were
prepared (ADH 0.1~ w/w; Lactose 99.9% w/w). We find that
optimisation of the spray-drying step is required to
maximise the retention of enzyme activity. The general
conditions of Example 1 were used, but the inlet and outlet
temperature were changed to give conditions which allowed
us to produce microparticles of the desired size (4-5 ~m)
that retained full activity after drying and reconstitution
in aqueous media. The percentage of activity retained
compared with the original material is shown for each of
the spray-drying conditions shown (Table 3). The
microcapsules were smooth and spherical and contained air
as evidenced by their appearance in diphenylxylene (DPX)
under light microscopy.
Table 3
Run Inlet Temp. Outlet Temp.Activity
C C Remaining
(%)
1 220 73 57
2 175 71 98
Exam~le 4
A series of experiments was performed under the
conditions described in Example 1, to examine the influence
of liquid feed rate on the yield of intact spherical
particles. We find that, using the ability of gas-
containing microparticles to reflect ultrasound, we are
able to determine optimal condition for maximising the
yield of intact smooth spherical microcapsules. The
microparticles formed after spray-drying are heat-fixed, to
render them insoluble, and then suspended in water to make
the echo measurements. We find that increasing the liquid
feed rate decreases the number of intact microparticles
formed during the initial spray-drying (Table 4). The mean
particle size and overall pressure stability, i.e.
WO96/09814 ~ /~b55/02279
18 0 ~ 1 9 ~ 4
thickness of the shell, do not change but the total
echogenicity does, as the liquid flow rate is increased
from 4 to 16 ml/min. We find that slower rates of
evaporation (at higher liquid flow rates) lead to fewer intact spherical particles being formed.
Table 4
Flow Rates (ml/min) 4 8 12 16
Mean size (~m) 3.08 3.04 3.13 3.07
Echogenicity (video 22 21 14 10
density units)
Echogenicity after 20 18 10 8
pressure (video density
units)
The assay was conducted by resuspending the heat-fixed
microparticles at a concentration of lx106 ml in 350 ml of
water. This solution is stirred slowly in a 500 ml beaker
above which is mounted an 3.5 MHz ultrasound probe attached
to a Sonus 1000 medical imaging machine. The grey scale
images obtained are captured by an image analyser and
compared against a water blank to yield video density units
of echo reflectance. The assay can also be adapted to
~x~;ne the pressure resistance, by assessing the echo-
reflectance before and after exposure of the sample to
cyclical bursts of pressure applied to the stock solution
of particles. This analysis distinguishes incomplete
particles which entrain air upon reconstitution, from fully
spherical particles which Uencapsulate" air within the
shell. Incomplete particles do not show pressure
resistance and lose the ability to reflect ultrasound
; ^~iately. The dose response for fixed albumin particles
of Example 1 is c. 5, 9, 13, 20, 22 and 24 VDU's
(backscatter intensity) at respective microcapsule
concentrations of 0.25, 0.5, 1, 2, 3 and 4 x 10 per ml.5 Exam~le 5
Significant experimentation to reduce the particle
size and narrow the size distribution has been performed.
This was pursued to effectively increase the gas content of
WO96/09814 P~ 5S~J7~
19 2 1 9 9 9 5 4
the echocontrast agent and reduce the number of oversized
particles. This exercise is also beneficial to respiratory
formulations in that it maximises the potential number of
respirable particles in the l-5 ~m range and produces
inherently more smooth particles which will be less
cohesive than non-spherical particles of similar size.
We find that it is possible to reduce particle size by
lowering the solutes content of the feedstock. This
effect, is in part, mediated by the effects of viscosity on
droplet formation. However, we also find that by lowering
the solute content under the conditions we use leads to a
significant decrease in the number of intact particles. By
further experimentation we have found that incorporation of
water-miscible, volatile solvents in the feedstock,
significantly increase the rate of shell formation during
drying, with a concomitant increase in the number of intact
particles or hollow particles (Table 5). The assessment of
hollowness is made by microscopic evaluation of particles
floating to the surface of the coverslip on a
haemocytometer versus particle count by Coulter counting.
Table 5
Run HSA ContentEthanol Mean Percentage
ofContent of Particle of Hollow
Feedstock Feedstock Size Particles
(%) (%) (~m) (%)
l lO 0 3.7 12.5
2 lO 25 3.52 64.3
ExamPle 6
A range of materials has been used to manufacture
smooth spherical soluble microparticles. The range Of
microparticles produced includes inert materials such as
HSA, lactose, mannitol, sodium alginate; active materials
alone such as ~l-antitrypsin; and mixtures of active and
inert carrier such as lactose/alcohol dehydrogenase,
lactose/budesonide, HSA/salbutamol. In all cases, smooth,
spherical and gas-containing particles are produced.
WO96/09814 PCT/~b~S,'~9
2 ~ 9 ~ ~ ~ 4
We have assessed the success of the process in
maintaining control over the morphology of the particles.
The particles are suspended in propanol and then visualised
by microscopy. Those particles which contain gas appear to
have an intense white core surrounded by an intact black
rim whilst broken or miss-formed particles appear as
ghosts. Microscopic evaluation of the following
microparticles exemplifies the range of materials and
actives which can be dried to produce smooth spherical
particles:
HSA
Casein
Haemoglobin
Lactose
ADH/lactose
HSA/Peroxidase
Lactose/Salbutamol
Lactose/Budesonide
Example 7
Lactose and Budesonide were spray-dried under the
conditions described in the table below (Table 6).
Table 6
Parameter Setting
Inlet Temperature 220C
Outlet Temperature 85C
Atomisation Pressure 7.5 bar
Damper Setting 0.5
Feed Rate 3.88 g/min
Stock Solution 9.3% w/v Budesonide, 85%v/v
Ethanol 19% w/v lactose
The resultant dry powder was blended with excipient
grade lactose in a V type blender in the proportions
outlined in Table 7. The blends were then loaded into
gelatin capsules and discharged from a Rotahaler into a
twin stage impinger run at 60 l/min. The respirable
fraction was calculated as the percentage deposited into
the lower chamber.
wog6/09814 ~ ~ 1 9 9 ~ glG~95l02179
21
Table 7
Formulation % Budesonide % spray % fast flow Respirable
Number in spray dried lactose in fraction
dried product in blend
particle~ blend
1 9.3 10 90 42
2 9.3 15 85 29
3 9.3 20 80 34
4 S.~ 30 70 36
The respirable fractions obtained are considerably
superior to micronised product currently used in this
device which are usually in the range of 10-20% maximum.
The BudesonidelLactose formulations detailed in
Example 7 were tested in an experimental gravity fed multi-
dose DPI. The parameters examined were the variation of
emitted dose over 30 shots and the respirable fraction in
a four-stage impinger device. The results are shown below
(Table 8).
Table 8
Formulation Dose Fine Particle CofV on
20 Number (mg) (Respirable) Emitted
Fraction (%) Dose (%)
1 4 52 2.0
2 4.2 42 2.8
3 3.7 S8 8.1
For current DPI devices, the preliminary US
Pharmacopoeia recommendation appears likely to be less than
2S% variation in the emitted dose. Clearly in all of the
formulations tested so far we are within the current
specifications and in the case of formulations 1 and 2 we
are significantly under the current limits.
Exam~le 8
To decrease the dissolution rate of soluble
microcapsules as described in preceding Examples,
microcapsules may be coated with fatty acids such as
3S palmitic or behenic acids. The soluble microcapsules of
WO96/09814 P~-l/~b55~2J9
22 2 ~ 9 ~ ~ 5 ~
Example 1 were coated by suspending a mixture of soluble
HSA microcapsules and glucose (50% w/w) in an ethanolic
solution containing 10% palmitic or behenic acid. The
solution was evaporated and the resultant cake milled by
passage through a Fritsch mill.
The efficacy of coating was assessed by an indirect
method derived from our previous ultrasound studies.
Ultrasound images were gathered from a beaker of water
containing 1 x 106 microcapsules/ml using a HP Sonus 1000
ultrasound machine linked to an image analyser. Video
intensity over a blank reading (VDU~ was measured over time
(Table 9).
The uncoated microcapsules very quickly lost all air
and thus the potential to reflect ultrasound. However,
coated microcapsules retained their structure for a longer
period and hence showed a prolonged signal over several
minutes.
Table g
Echogenicity of Coated HSA Microcapsules
20Time (min) Ec-ogenicity (VD )
HSA only HSA/Palmitic HSA/Behenic
Coated Coated
0 1.75 1.91 0.88
0.043 0.482 0.524
0 0 0.004
Exam~le g
Soluble mannitol microcapsules were prepared as set
out in Example 1 (15% aqueous mannitol spray-drying
feedstock) and coated with palmitic acid and behenic acid
as described in Example 8. A sample of each was suspended
in water and the echogenicity measured. Ten minutes after
the initial analysis, the echogenicity of the suspended
samples was repeated (Table 10).
W096/09814 0 2 1 9 9 9 ~ ~ GB95/o227s
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Table 10
Echogenicity of Coated Mannitol Microcapsules
r ' Time Echogenicity (VD )
(min)Mannitol + Palmitic + Behenic
- 5 o 1.6 1.7 0.92
0.33 0.5 0.24
17 00.84 o
Exam~le 10
Soluble microcapsules with a model active (Lysine-
Fluoroscein) contained within the matrix were prepared to
allow the production of a free-flowing dry powder form of
the active~ compound. On dissolution of the
microcapsules, the active compound was released in its
native form.
Using lysine as a model compound, the molecule was
tagged with fluorescein isothiocyanate (FITC) to allow the
compound to be monitored during the preparation of the
soluble microcapsules and the subsequent release during
dissolution.
3 g of lysine was added to FITC (0.5 g total) in
carbonate buffer. After one hour incubation at 30C, the
resultant solution was tested for the formation of the
FITC-lysine adduct by TLC. This showed the presence of a
stable FITC-lysine adduct.
The FITC-lysine adduct was mixed with 143 ml of 25%
ethanol containing 100 mg/ml HSA to give the spray-drying
feedstock. The spray-drying conditions used to form the
microcapsules are detailed in Table 11 below. In the
absence of ethanol we ha~e found that only a small
percentage of the particles are smooth and spherical.
The spray-drying process produced 17.21 g of
microcapsules that did not dissolve when a sample was
resuspended in ethanol. Moreover, no release of the FI~C-
lysine adduct was observed. However, when 10 ml water wasadded to the ethanol-suspended microcapsules, the
W096/09814 PCTI~bg5/~/9
24 219~
microcapsules dissolved and the FITC-lysine was released.
Analysis of the adduct using TLC before incorporation into
the microcapsules and after release from the microcapsules
on dissolution showed the model compound was unch~ed.
Table 11
Spray-Drying Conditions
Parameter Setting
Inlet Temperature 220C
Outlet Temperature 850C
Atomisation Pressure 7.5 bar
Damper Setting 0.5
Feed ~ate 3.88 g/min
Stock Solution 25% v/v Ethanol, 10% w/v HSA
The soluble microcapsules were sized in a non-aqueous
system of ammonium thiocyanate and propan-2-ol using a
Multisizer II (Coulter Electronics). The microcapsules had
a mean size of 3.28 + 0.6 ~m and with 90% of the mass
within 2-5 ~m.
The microcapsules were mixed with glucose (50% w/w
microcapsules : 50% w/w glucose), and milled by the passage
of the mixture through a Fritsch mill three times. When a
sample of the powder was added to water, the FITC-lysine
was released intact when compared with its original form as
determined by TLC analysis. This example shows the
feasibility of making an amino acid or peptide formulation
which could be used for respiratory formulations, which
incorporates HSA within the formulation.
Example 11
500 mg beclomethasone was dissolved in ethanol and
added to 50 ml HSA feedstock (10% w/v~ and spray-dried
using the conditions outlined in Example 10. The
microcapsules hence formed were sized in the non-aqueous
system as detailed in Example 10. The microcapsules had a
mean size of 3.13 + 0.71 ~m, 90~ of which were between 2
and 5 ~m.
WO96/09814 25 0 2 1 9 9 9 5CT4/GBg5/02279
The beclomethasone was extracted from the
microcapsules by the precipitation of the HSA in 10~ TCA,
and the supernatant was extracted into ethanol. The
ethanol extract was analysed using HPLC, at a wavelength
242 nm. The beclomethasone detected in this extract exists
in the free state, but when the albumin pellet was
extracted the presence of beclomethasone bound to native
HSA was observed. It was found that although the majority
of the active compound was in the free state, some was
present in the albumin-bound state. Since albumin
partitions only slowly into the bloodstream, this allows
control over the release of the active compound over an
extended period of time, compared to free drug.
ExamPle 12
Whereas in Examples 10 and 11 at least, any binding of
the active compounds was an effect of the intrinsic nature
of albumin, this Example gives a product following initial
cross-linking of the active compound, prior to spray-
drying.
To a 10 mg/ml solution of methotrexate, 25 mg
carbodiimide (EDCI) was added. The solution was stirred
for 4 hours to initiate and ensure complete activation of
the methotrexate. 50 mg HSA was added to the activated
drug and stirred for 3 hours at room temperature. The
methotrexate is chemically bound to the HSA via the amine
groups on the albumin. This conjugate was then used as the
spray-drying feedstock as detailed in Example 10.
The soluble microcapsules thus made were sampled,
characterised and analysed for drug content. The
microcapsules had a mean size of 3.2 + 0.6 ~m with 90% by
mass between 2-5 ~m. The analysis of the drug content of
the microcapsules showed that the microcapsules did not
release drug; even after dissolution, drug was still bound
to the HSA. Proteinase K digestion of the albumin released
the bound drug which was shown to be linked to only a
limited number of amino-acids and small peptides. It has
been shown previously that the activity of doxorubicin
WO96/09814 PCT/~bS5~279
021 99 ~4
26
bound to polymeric carriers proves beneficial in tumours,
showing the multidrug-resistant phenotype.
Example 13
Naproxen microcapsules were prepared as detailed in
Examples 10 and 12 using a ratio of 1 to 5, drug to HSA.
The soluble microcapsules retained the active compound of
a non-aqueous solvent. Moreover, on dissolution of the
microcapsules in aqueous solution, the active compound was
still bound to the albumin, as shown by HPLC analysis at
262 nm, as before. The naproxen was released from the
albumin on digestion with proteinase K and esterases.
ExamPle 14
Using samples of the microcapsules produced in
Examples 8 to 13, an assessment of their behaviour in a dry
powder inhaler was made. The dosing reproducibility of
each formulation was assessed in conjunction with the
aerolisation of the sample by microscopic evaluation.
A sample of each formulation was added to the storage
funnel of an experimental dry powder inhaler (DPI). The
dry powder inhaler used pressurised air to force the powder
into a dosing measure. The dosing measure used was
calibrated using spray-dried lactose.
Although the amounts dispensed into the dosing measure
varied between samples as a function of their composition,
the dosing reproducibility for each sample was very
consistent; with a mean of 5.0 + 0.25 mg obtained for three
dosing trials.
The aerolisation behaviour of the samples was tested
by connecting the inhaler to a vacuum ch~h~r; simulated
inhalation was achieved by the release of the vacuum
through the DPI and collection of the airborne dose was
made on resin coated microscope slides. These slides were
evaluated for dispersion of the particles. The slides
showed that the DPI had deagglomerated the samples forming
an even dispersion of microparticles on the microscope
slides.
WO96/09814 PCT/~95~7~
27 0 2 1 9 9 9 5 4
ExamPle 15
The performance of the dry powder formulations from
Examples lO to 13 was analysed using the twin impinger
method (Apparatus A for pressurised inhalations, British
Pharmacopoeia 1988) following discharge from a Rotahaler
(Glaxo UK) with 7 ml in stage l and 30 ml in stage 2 of
distilled water. The formulations were delivered from size
3 gelatin capsules using a Rotahaler attached to the twin
impinger using a rubber adapter. The vacuum pump was
operated at 60 l/min for two 3 second bursts. The amount
of each sample reaching stage l and stage 2 levels of the
impinger was analysed. All samples showed the largest
percentage deposition to occur in stage 2 of the impinger
indicating optimal sized particles for alveoli delivery.
Exam~le 16
A comparison of the dosing and deposition of fixed
insoluble microcapsules and soluble microcapsules as
produced in Example lO was made in the lung of rabbits.
Anaethestised New Zealand white rabbits were dosed
either with soluble microcapsules or fixed microcapsules.
The dosing was carried out using a computer controlled
nebuliser (Mumed Ltd., UK). The soluble microcapsules were
suspended in CFC ll and the fixed particles were suspended
in water. After dosing, the lungs of the rabbits were
removed and an assessment of the deposition of the capsules
made.
The fixed capsules were found intact in the alveoli
tissue of the lung. This showed that the microcapsules
were of the appropriate size for dispersion through the
lungs. In comparison, no evidence of the presence of
intact soluble microcapsules was found, the càpsules having
_ dissolved in the fluids of the lung. However, the presence
of FITC-lysine adduct was observed in some of the alveoli
tissue when studied using fluorescent microscopy. In
addition, the presence of the adduct was also found the
blood and urine of the animals, as opposed to that of the
fixed capsules which showed no presence in either.
