U.S. patent application number 10/804679 was filed with the patent office on 2004-09-09 for spray-dried microparticles and their use as therapeutic vehicles.
Invention is credited to Heath, David, Johnson, Richard A., Senior, Peter J., Sutton, Andrew D..
Application Number | 20040175328 10/804679 |
Document ID | / |
Family ID | 31981936 |
Filed Date | 2004-09-09 |
United States Patent
Application |
20040175328 |
Kind Code |
A1 |
Sutton, Andrew D. ; et
al. |
September 9, 2004 |
Spray-dried microparticles and their use as therapeutic
vehicles
Abstract
Microparticles of a water-soluble material, which are smooth and
spherical, and at least 90% of which have a mass median particle
size of 1 to 10 .mu.m, and which carry a therapeutic or diagnostic
agent can successfully be used in dry powder inhalers to deliver
the said agent.
Inventors: |
Sutton, Andrew D.;
(Nottingham, GB) ; Johnson, Richard A.;
(Nottingham, GB) ; Senior, Peter J.; (Derbyshire,
GB) ; Heath, David; (Nottingham, GB) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
31981936 |
Appl. No.: |
10/804679 |
Filed: |
March 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10804679 |
Mar 18, 2004 |
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09382561 |
Aug 25, 1999 |
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6709650 |
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09382561 |
Aug 25, 1999 |
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08487420 |
Jun 7, 1995 |
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5993805 |
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08487420 |
Jun 7, 1995 |
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07956875 |
Mar 15, 1993 |
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5518709 |
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Current U.S.
Class: |
424/9.32 ;
424/491 |
Current CPC
Class: |
A61K 9/1694 20130101;
B01J 13/04 20130101; A61K 9/5089 20130101; A61K 49/00 20130101;
A61K 9/0075 20130101; Y10S 514/965 20130101; A61K 49/223 20130101;
B01J 13/14 20130101; B01J 13/043 20130101; A61K 9/1688 20130101;
A61K 49/225 20130101 |
Class at
Publication: |
424/009.32 ;
424/491 |
International
Class: |
A61K 049/00; A61K
009/16; A61K 009/50 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 20, 1994 |
EP |
94307126.6 |
Apr 10, 1991 |
GB |
9107628.1 |
Claims
What we claim is:
1. Microparticles of a water-soluble material, which are smooth and
spherical, and at least 90% of which have a mass median particle
size of 1 to 10 .mu.m.
2. Microparticles according to claim 1, wherein said particle size
is 1 to 5 .mu.m.
3. Microparticles according to claim 1, which have a maximum
interquartile range of 3 .mu.m.
4. Microparticles according to claim 3, which have a maximum
interquartile range of 2 .mu.m.
5. Microparticles according to claim 1, which are sterile.
6. Microparticles according to claim 1, which are at least partly
coated with a water-insoluble material.
7. Microparticles according to claim 1, which additionally carry a
receptor-binding component.
8. Microparticles according to claim 1, wherein said water-soluble
material is a carbohydrate.
9. Microparticles according to claim 1, wherein said water-soluble
material is an amino- or polyamino-acid.
10. Microparticles according to claim 1, wherein said water-soluble
material is a fatty acid or ester thereof.
11. Microparticles-according to claim 1, wherein said water-soluble
material is a protein, peptide or enzyme.
12. Microparticles according to claim 1, wherein said water-soluble
material is a human protein or fragment or recombinant thereof.
13. Microparticles according to claim 12, wherein said protein is
human serum albumin.
14. Microparticles according to claim 1, wherein said water-soluble
material is chemically or enzymatically modified, prior to
formation of the microparticles.
15. Microparticles according to claim 1, which carry a diagnostic
agent.
16. Microparticles according to claim 1, which carry a therapeutic
agent.
17. Microparticles according to claim 15 or claim 16, obtainable by
spray-drying an aqueous solution of said water-soluble material and
said therapeutic or said diagnostic agent.
18. An inhaler device adapted to deliver a therapeutic agent via
the pulmonary airways, which comprises said therapeutic agent in
the form of microparticles according to claim 16 or claim 17.
19. In a method of treating a complaint by administration to the
patient of an effective amount of a therapeutic agent that acts via
pulmonary airways to treat the complaint, the improvement
comprising administration of said therapeutic agent in the form of
microparticles according to claim 16 or claim 17.
Description
REFERENCE TO EARLIER APPLICATION
[0001] This Application is a continuation-in-part of application
Ser. No. 07/956,875 filed Mar. 11, 1993.
FIELD OF THE INVENTION
[0002] 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.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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.
[0005] 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 .mu.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.
[0006] Current methods of aerosolising drugs for inhalation include
nebulisation, metered dose inhalers and dry powder systems.
Nebulisation of aqueous solutions requires large volumes of drugs
and involves the use of bulky and non-portable devices.
[0007] 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.
[0008] 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.
[0009] 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 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.
[0010] 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 .mu.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.
[0011] 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. WO-A-91/12823 (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 .mu.m diameter. WO-A-9/09629
discloses liposomes for use as ultrasound contrast agents.
[0012] 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
microspheres was apparently obtained, as a further sieving step was
necessary.
[0013] 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 .mu.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.
[0014] 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 .mu.m in size. No
directions are given as to what conditions should be used, in order
to obtain such a product.
[0015] 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.
[0016] Conte et al (1994) Eur. J. Pharm. Biopharm. 40(4):203-208,
disclose spray-drying from aqueous solution, with a 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.
[0017] 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
[0018] 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, the wall-forming material is
substantially unaffected by spray-drying. Thus, highly uniform
microparticles, microspheres or microcapsules of heat-sensitive
materials such as enzymes, peptides and proteins, e.g. HSA, and
other polymers, may be prepared and formulated as dry powders.
[0019] By contrast to the prior art, it has 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 .mu.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 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 .mu.m on a
number basis, within an interquartile range of 2 .mu.m, and with
less than 0.5 .mu.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 1/min) resulted in excellent
separation of microparticles from excipient.
[0020] 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
polymer 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.
[0021] 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 .mu.m.
[0022] In essence therefore we have defined how to produce
microparticles which are: predominantly 1-5 .mu.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.
[0023] 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 across
cellular barriers to the systemic circulation and beyond.
[0024] 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. 10-30% 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.
[0025] 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
1.0-10.0.times.10.sup.5 Pa, preferably 2-8.times.10.sup.5 Pa, and
most preferably about 7.5.times.10.sup.5 Pa. Other parameters may
be varied as disclosed below. In this way, novel microspheres may
be obtained.
[0026] 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 within a 2 .mu.m range
and at least 90%, preferably at least 95% or 99%, have a diameter
within the range 1.0-8.0 .mu.m.
[0027] The interquartile range may be 2 .mu.m, with a median
diameter of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5 .mu.m.
[0028] Thus, at least 30%, 40%, 50% or 60% of the microspheres may
have diameters within the range 1.5-3.5 .mu.m, 2.0-4.0 .mu.m,
3.0-5.0 .mu.m, 4.0-6.0 .mu.m, 5.0-7.0 .mu.m or 6.0-8.0 .mu.m.
Preferably a said percentage of the microspheres have diameters
within a 1.0 .mu.m range, such as 1.5-2.5 .mu.m, 2.0-3.0 .mu.m,
3.0-4.0 .mu.m, 4.0-5.0 .mu.m, 5.0-6.0 .mu.m, 6.0-7.0 .mu.m or
7.0-8.0 .mu.m.
[0029] 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 .mu.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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Preferred aspects of the present invention will be
particularly described by way of example and with reference to the
accompanying drawings, in which:
[0031] FIGS. 1A and 1B are each overlay chromatograms of HSA before
(*) and after spray-drying, respectively from gel-permeation
chromatography and reverse-phase HPLC analysis of tryptic
digests;
[0032] FIG. 2 is a plot of volume with respect to size, showing the
size control of microparticles by manipulation of solute
concentration, from feedstocks containing 1) 15% w/v, 2) 12.5% w/v,
3) 10% w/v, and 4) 7.5% w/v HSA (analysis of fixed particles by
Coulter counting);
[0033] FIG. 3 is an ultrasound dose-response curve for
microparticles manufactured in accordance with Example 1; and
[0034] FIG. 4 is a partly cut-away perspective view from the front
and one side of suitable spray-drying apparatus for use in the
process of the invention.
DESCRIPTION OF THE INVENTION
[0035] 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 (U.S.
Pat. No. 4,888,398) or a polysaccharide such as polydextrose.
[0036] Generally, the wall-forming material can be selected from
most hydrophilic, biodegradable physiologically compatible
polymers. Among such polymers one can cite polysaccharides of low
water solubility, polylactides and polyglycolides and their
copolymers, copolymers of lactides and lactones such as
.epsilon.-capcrolactone, .delta.-valerolactone, polypeptides, and
proteins such as gelatin, collagen, globulins and albumins. Other
suitable polymers include poly-(ortho)esters (see for instance U.S.
Pat. No. 4,093,709, U.S. Pat. No. 4,131,648, U.S. Pat. No.
4,138,344, U.S. Pat. No. 4,180,646); polylactic and polyglycolic
acid and their copolymers, for instance Dexon (see J. Heller (1980)
Biomaterials 1, 51); poly(DL-lactide-co-.delta.-cap- rolactone),
poly(DL-lactide-co-.delta.-valerolactone),
poly(DL-lactide-co-.delta.-butyrolactone),
polyalkylcyano-acrylates; polyamides; polyhydroxybutyrate;
polydioxanone; poly-.beta.-aminoketones (Polymer 23 (1982), 1693);
polyphosphazenes (Science 193 (1976), 1214); and polyhydrides.
References on-biodegradable polymers can be found in R. Langer et
al (1983) Macromol. Chem. Phys. C23, 61-125. Polyamino-acids such
as polyglutamin and polyaspartic acids can also be used as well as
their derivatives, i.e. partial esters with lower alcohols or
glycols. One useful example of such polymers is
poly(t-butylglutamate). Copolymers with other amino-acids such as
methionine, leucine, valine, proline, glycine, alamine, etc. are
also possible. Recently some novel derivatives of polyglutamic and
polyaspartic acid with controlled biodegradability have been
reported (see WO-A-87/03891; U.S. Pat. No. 4,888,398 and
EP-A-130935 incorporated here by reference). These polymers (and
copolymers with other amino-acids) have formulae of the following
type:
--(NH--CHA--CO).sub.2 (NH--CHX--CO).sub.3
[0037] where X designates the side chain of an amino-acid residue
and A is a group of formula
--(CH.sub.2).sub.nCOOR.sup.1R.sup.2OCOR(II), with R.sup.1 and
R.sup.2 being H or lower alkyls, and R being alkyl or aryl; or R
and R.sup.1 are connected together by a substituted or
unsubstituted linking member to provide 5- or 6-membered rings.
[0038] A can also represent groups of formulae:
--(CH.sub.2).sub.nCOO--CHR.sup.1COOR (I)
[0039] and
--(CH.sub.2).sub.nCO(NH--CHX--CO).sub.mNH--CH(COOH)--CH.sub.3).sub.pCOOH
(II)
[0040] and corresponding anhydrides. In all these formulae n, m and
p are lower integers (not exceeding 5) and x and y are also
integers selected for having molecular weights not below 5000.
[0041] The aforementioned polymers are suitable for making the
microspheres according to the invention and, depending on the
nature of substituents R, R.sup.1, R.sup.2 and X, the properties of
the wall can be controlled, for instance, strength, elasticity and
biodegradability. For instance X can be methyl (alanine), isopropyl
(valine), isobutyl (leucine and isoleucine) or benzyl
(phenylalanine).
[0042] 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 (HA) derived from blood donations or,
ideally, from the fermentation of microorganisms (including cell
lines) which have been transformed or transfected to express
HA.
[0043] Techniques for expressing HA (which term includes analogues
and fragments of human albumin, for example those of EP-A-322094,
and polymers of monomeric albumin) are disclosed in, for example
EP-A-201239 and EP-A-286424. All references are included herein by
reference. "Analogues and fragments" of HA include all polypeptides
(i) which are capable of forming a microcapsule in the process of
the invention and (ii) of which a continuous region of at least 50%
(preferably at least 75%, 80%, 90% or 95%) of the amino-acid
sequence is at least 80% homologous (preferably at least 90%, 95%
or 99% homologous) with a continuous region of at least 50%
(preferably 75%, 80%, 90% or 95%) of human albumin. HA which is
produced by recombinant DNA techniques is particularly preferred.
Thus, the HA may be produced by expressing an HA-encoding
nucleotide sequence in yeast or in another microorganism and
purifying the product, as is known in the art. Such material lacks
the fatty acids associated with serum-derived material. Preferably,
the HA is substantially free of fatty acids; i.e. it contains less
than 1% of the fatty acid level of serum-derived material.
Preferably, fatty acid is undetectable in the HA.
[0044] In the following description of preferred embodiments, the
term "protein" is used since this is what we prefer, but it is to
be understood that other biocompatible wall-forming materials can
be used, as discussed above.
[0045] 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.
[0046] The preparation to be sprayed may contain substances other
than the wall-forming material and solvent or carrier liquid. Thus,
the aqueous phase may contain 1-20% by weight of water-soluble
hydrophilic compounds like sugars and polymers as stabilisers, e.g.
polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene
glycol (PEG), gelatin, polyglutamic acid and polysaccharides such
as starch, dextran, agar, xanthan and the like. Similar aqueous
phases can be used as the carrier liquid in which the final
microsphere product is suspended before use. Emulsifiers may be
used (0.1-5% by weight) including most physiologically acceptable
emulsifiers, for instance egg lecithin or soya bean lecithin, or
synthetic lecithins such as saturated synthetic lecithins, for
example, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl
choline or distearoyl phosphatidyl choline or unsaturated synthetic
lecithins, such as dioleyl phosphatidyl choline or dilinoleyl
phosphatidyl choline. Emulsifiers also include surfactants such as
free fatty acids, esters of fatty acids with polyoxyalkylene
compounds like polyoxypropylene glycol and polyoxyethylene glycol;
ethers of fatty alcohols with polyoxyalkylene glycols; esters of
fatty acids with polyoxyalkylated sorbitan; soaps;
glycerol-polyalkylene stearate; glycerol-polyoxyethylene
ricinoleate; homo- and copolymers of polyalkylene glycols;
polyethoxylated soya-oil and castor oil as well as hydrogenated
derivatives; ethers and esters of sucrose or other carbohydrates
with fatty acids, fatty alcohols, these being optionally
polyoxyalkylated; mono-, di- and triglycerides of saturated or
unsaturated fatty acids, glycerides or soya-oil and sucrose.
[0047] Additives can be incorporated into the wall of the
microspheres to modify the physical properties such as
dispersibility, elasticity and water permeability.
[0048] Among the useful additives, one may cite compounds which can
"hydrophobise" the wall in order to decrease water permeability,
such as fats, waxes and high molecular weight hydrocarbons.
Additives which improve dispersibility of the microspheres in the
injectable liquid-carrier are amphipathic compounds like the
phospholipids; they also increase water permeability and rate of
biodegradability.
[0049] Additives which increase wall elasticity are the
plasticisers like isopropyl myristate and the like. Also, very
useful additives are constituted by polymers akin to that of the
wall itself but with relatively low molecular weight. For instance
when using copolymers of polylactic/polyglycolic type as the
wall-forming material, the properties of the wall can be modified
advantageously (enhanced softness and biodegradability) by
incorporating, as additives, low molecular weight (1000 to 15,000
Dalton) polyglycolides or polylactides. Also polyethylene glycol of
moderate to low Mw (e.g. PEG 2000) is a useful softening
additive.
[0050] The quantity of additives to be incorporated in the wall is
extremely variable and depends on the needs. In some cases no
additive is used at all; in other cases amounts of additives which
may reach about 20% by weight of the wall are possible.
[0051] 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 .mu.m diameter. These
figures refer to at least 90% of the population of microcapsules,
the diameter being measured with a Coulter Master Sizer II. 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" (Regd..TM.) are not formed.
[0052] 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. preferably 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 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 100.degree. C., preferably
at least 110.degree. C., is generally sufficient to ensure
hollowness and the temperature may be as high as 250.degree. C.
without the capsules bursting. About 180-240.degree. C., preferably
about 210-230.degree. C. and most preferably about 220.degree. C.,
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
40-150.degree. C. 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.
[0053] The microcapsules comprise typically 96-98% monomeric
HA.
[0054] More particularly, microparticles of the invention
preferably have a maximum interquartile range of 3 .mu.m, more
preferably 2 .mu.m, and most preferably 1.5 .mu.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.
[0055] 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.sup.2.d).sup.a+B.(M.sub.air/M.sub.liq).sup.-b
[0056] Where D=Mean droplet size
[0057] A=Constant related to nozzle design
[0058] B=Constant related to liquid viscosity
[0059] V=Relative air velocity between liquid and nozzle
[0060] d=Air density
[0061] M.sub.air and M.sub.liq=Mass of air and liquid flow a and
b=Constants related to nozzle design
[0062] 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
.mu.m. For the production of microparticles in the size range
described herein we use an air to liquid ratio ranging from
20-1000. 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.
[0063] The amount of the 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.
[0064] 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 .alpha.-1 anti-trypsin, or 0.1 mg/day of an active material
such as beclomethasone.
[0065] 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.
[0066] 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 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.
[0067] 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.
[0068] 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.
[0069] A further embodiment of the invention is the co-spray-drying
of the active principle with HSA in order to facilitate
stabilisation of the active principle during formulation, packing
and, most importantly, during 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 endothelial lining.
[0070] 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.
[0071] 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.
[0072] 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 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.
[0073] The following Examples illustrate the invention. The spray
dryer used in the Examples and shown in FIG. 4 is available from
A/S Niro Atomizer, Soeborg, Denmark, under the trade name "Mobile
Minor". Details of its construction will now be given.
[0074] FIG. 4 shows a feeding device 1 and a ceiling air dispenser
2 which ensures effective control of the air flow pattern. Swirling
air is directed around the vaned disc atomiser. 3 is a rotary
atomiser or nozzle atomiser. 4 shows a stainless steel
interconnecting pipe system which can easily be stripped down for
cleaning. 5 are steps for access to the chamber top. 6 is the
switch for an air valve for activation of the pneumatic lifting
device when raising the chamber lid. 7 is a highly-efficient
stainless steel cyclone in which the powder and the exhausted
drying air are separated. 8 is a glass jar in which the powder is
recovered. 9 is a centrally located instrument panel. 10 is a
centrifugal exhaust fan with three-phase motor. 11 is a damper for
air flow control and 12 is an electric air heater which provides
drying air temperatures up to 350.degree. C. The drying air
temperature can be continuously adjusted using a percentage timer
switch. The maximum powder consumption is 7.6 kW.
1 Evaporative capacity Inlet Air Outlet Air Evaporative Drying Air
Temperature Temperature Capacity 85 kg/h 150.degree. C. 80.degree.
C. 1.3 kg/h 85 kg/h 170.degree. C. 85.degree. C. 1.7 kg/h 80 kg/h
200.degree. C. 90.degree. C. 2.5 kg/h 80 kg/h 240.degree. C.
90.degree. C. 3.4 kg/h 75 kg/h 350.degree. C. 90.degree. C. 7.0
kg/h
[0075]
2 Weight and dimension Weight 280 kg Length 1800 mm Height 2200 mm
Width 925 mm
[0076] Power
[0077] The unit can only be operated on a three-phase power supply
(50 or 60 Hz) at alternative voltages of 440, 415, 400, 380, 220,
220 V.
[0078] The "Mobile Minor" comprises a centrifugal atomizer (Type
M-02/B Minor), driven by an air turbine at an air pressure of min 4
bar and up to max 6 bar. At 6 bar an atomizer wheel speed of approx
33,000 rpm is reached. Turning on and off the compressed air to the
atomizer is done by means of a valve placed in the instrument
panel. The maximum consumption of compressed air to the atomizer is
17 Nm3/h at a pressure of 6 bar. All parts coming into contact with
the liquid feed and powder are made of stainless steel AISI 316,
except for the pump feed tube and the atomizer wheel, which is made
of stainless steel AISI 329, made to resist high centrifugal
force.
[0079] The drying chamber has an inside made of stainless steel
AISI 316, well insulated with Rockwool, and covered outside with a
mild steel sheeting. The drying chamber is provided with a side
light and observation pane for inspection during the operation. The
roof of the drying chamber is made inside of stainless steel AISI
316 and outside of stainless steel AISI 304.
[0080] An air disperser made of stainless steel AISI 304 is used
for distribution of the air in the drying chamber in order to
achieve the best possible drying effect. An air duct, made of
stainless steel AISI 316, provides lateral transportation of the
exhaust air and the powder to the cyclone, which is made of
stainless steel AISI 316 and designed to separate the powder and
air.
[0081] A closing valve of the butterfly valve type, also made of
stainless steel AISI 316 and having a gasket of silicone rubber, is
used for powder discharge under the cyclone into a powder
collecting glass jar tightly placed under the cyclone by means of a
spring device.
[0082] A fan made of silumin, complete with 3-phase squirrel-cage
motor, 0.25 kW, and V-belt drive with belt-guard, draws air and
powder through the drying chamber and cyclone.
[0083] An air heater heats the drying air by means of electricity
(total consumption 7.5 kWh/h, infinitely variable) and can give
inlet air temperatures of up to about 350.degree. C., although this
is generally too high for preparing the microcapsules of the
invention.
[0084] Equipment for two-fluid nozzle atomisation may be added,
which is made of stainless steel AISI 316, consisting of entrance
pipe with nozzle holder, to be placed in the ceiling of the drying
chamber. The equipment includes an oil/water separator, reduction
valve and pressure gauge for compressed air to the two-fluid
nozzle. Consumption of compressed air: 8-15 kg/h at a pressure of
0.5-2.0 bar (0.5-2.0.times.10.sup.5 Pa).
[0085] A suitable feed pump for transport of wall-forming
preparation feed to the atomizer device is a peristaltic pump. The
pump is provided with a motor (1.times.220V, 50 Hz, 0.18 kW) and a
continuously variable gear for manual adjustment. A feed pipe made
of silicone hose leads from a feed tank (local supply) through the
feed pump to the atomization device. An absolute air filter,
consisting of prefilter, filter body in stainless steel and
absolute air filter, is used for the treatment of the ingoing
drying air to render it completely clean.
EXAMPLE 1
[0086] 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 atomizer 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 220.degree. C. the outlet air temperature was
maintained at 95.degree. C.
[0087] Compressed air was supplied to the two fluid atomising
nozzle at 2.0-6.0 Bar (2.0-6.0.times.10.sup.5 Pa). In this range
microcapsules with a mean size of 4.25-6.2 .mu.m are obtained.
[0088] Typically an increase in mean particle size (by reduced
atomisation pressure) led to an increase in the amount of
microcapsules over 10 .mu.m in size (see Table 1).
3TABLE 1 Effects of Atomisation Pressure on Frequency of
Microcapsules Over 10 .mu.m in Diameter Atomisation Pressure
(.times.10.sup.5 Pa) % Frequency over 10 mm 6.0 0.8 5.0 3.3 3.5 6.6
2.5 8.6 2.0 13.1
[0089] Under the conditions described, with a nozzle pressure of
7.5 bar, microparticles were produced with a particle size of 4.7
.mu.m. These soluble microparticles were smooth and spherical with
less than 1% of the particles over a particle size of 6 .mu.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 original and spray-dried HSA are
shown (FIG. 1). 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
(FIG. 1). Both analysis show, that under the conditions of
spray-drying described to produce microparticles of 4.7 .mu.m,
little or no structural damage is done to the protein.
EXAMPLE 2
[0090] Alpha-1 antitrypsin derived from human serum was spray-dried
under conditions similar to Example 1 with an inlet temperature of
150.degree. C. and an outlet temperature of 80.degree. C. 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
.mu.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 retention of inhibitory activity had been
achieved.
4 TABLE 2 Percentage of Activity Run Number Retained 1 84 2 222 3
148
EXAMPLE 3
[0091] 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
.mu.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.
5 TABLE 3 Activity Inlet Temp. Outlet Temp. Remaining Run .degree.
C. .degree. C. (%) 1 220 73 57 2 175 71 98
EXAMPLE 4
[0092] 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. 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.
6 TABLE 4 Flow Rates (ml/min) 4 8 12 16 Mean size (.mu.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)
[0093] The assay was conducted by resuspending the heat-fixed
microparticles at a concentration of 1.times.10.sup.6 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 examine 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 "encapsulate" air within the shell. Incomplete particles do
not show pressure resistance and lose the ability to reflect
ultrasound immediately. The dose response for fixed albumin
particles of Example 1 is shown in FIG. 3.
EXAMPLE 5
[0094] 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 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 1-5 .mu.m range and
produces inherently more smooth-particles which will be less
cohesive than non spherical particles of similar size.
[0095] We find that it is possible to reduce particle size by
lowering the solutes content of the feedstock (FIG. 2). 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.
7TABLE 5 HSA Content Ethanol Mean Percentage of Content of Particle
of Hollow Feedstock Feedstock Size Particles Run (%) (%) (.mu.m)
(%) 1 10 0 3.7 12.5 2 10 25 3.52 64.3
EXAMPLE 6
[0096] 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
.alpha.1-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. We have assessed the success of the process
in maintaining control over the morphology of the particles
produced by microscopy. 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.
[0097] HSA
[0098] Casein
[0099] Haemoglobin
[0100] Lactose
[0101] ADH/lactose
[0102] HSA/Peroxidase
[0103] Lactose/Salbutamol
[0104] Lactose/Budesonide
EXAMPLE 7
[0105] Lactose and Budesonide were spray-dried under the conditions
described in the table below (Table 6).
8 TABLE 6 Parameter Setting Inlet Temperature 220.degree. C. Outlet
Temperature 85.degree. C. 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
[0106] 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.TM. into a twin stage impinger run at 60 1/min.
The respirable fraction was calculated as the percentage deposited
into the lower chamber.
9TABLE 7 % Budesonide % spray in spray dried % fast flow
Formulation dried product in lactose in Respirable Number particles
blend blend fraction 1 9.3 10 90 42 2 9.3 15 85 29 3 9.3 20 80 34 4
5.7 30 70 36
[0107] 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.
[0108] The Budesonide/Lactose 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).
10 TABLE 8 Fine Particle CofV on Formulation Dose (Respirable)
Emitted Number (mg) Fraction (%) Dose (%) 1 4 52 2.0 2 4.2 42 2.8 3
3.7 58 8.1
[0109] For current DPI devices the US Pharmacopoeia states less
than 10% 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.
EXAMPLE 8
[0110] To decrease the dissolution rate of soluble microcapsules as
described in preceding Examples, microcapsules may be coated with
fatty acids such as palmitic or behenic acids. The soluble
microcapsules of 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.
[0111] 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.times.10.sup.6
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).
[0112] 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.
11TABLE 9 Echogenicity of Coated HSA Microcapsules Echogenicity
(VDU) HSA/Palmitic HSA/Behenic Time (min) HSA only Coated Coated 0
1.75 1.91 0.88 5 0.043 0.482 0.524 10 0 0 0.004
EXAMPLE 9
[0113] 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).
12TABLE 10 Echogenicity of Coated Mannitol Microcapsules Time
Echogenicity (VDU) (min) Mannitol + Palmitic + Behenic 0 1.6 1.7
0.92 10 0.33 0.5 0.24 17 0 0.84 0
EXAMPLE 10
[0114] 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.
[0115] 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.
[0116] 3 g of lysine was added to FITC (0.5 g total) in carbonate
buffer. After one hour incubation at 30.degree. C., 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.
[0117] 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 have found that
only a small percentage of the particles are smooth and
spherical.
[0118] 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 FITC-lysine adduct was observed.
However, when 10 ml water was added to the ethanol-suspended
microcapsules, the 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 unchanged.
13TABLE 11 Spray-Drying Conditions Parameter Setting Inlet
Temperature 220.degree. C. Outlet Temperature 85.degree. C.
Atomisation Pressure 7.5 bar Damper Setting 0.5 Feed Rate 3.88
g/min Stock Solution 25% v/v Ethanol, 10% w/v HSA
[0119] 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
{overscore (n)} 0.6 .mu.m and with 90% of the mass within 2-5
.mu.m.
[0120] 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
[0121] 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 no-aqueous system as detailed in Example 10. The
microcapsules had a mean size of 3.13.+-.0.71 .mu.m, 90% of which
were between 2 and 5 .mu.m.
[0122] 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
[0123] 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.
[0124] 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.
[0125] The soluble microcapsules thus made were sampled,
characterised and analysed for drug content. The microcapsules had
a mean size of 3.2.+-.0.6 .mu.m with 90% by mass between 2-5 .mu.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 bound to
polymeric carriers proves beneficial in tumours showing the
multidrug-resistant phenotype.
EXAMPLE 13
[0126] 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
[0127] To decrease the dissolution rate of soluble HSA
microcapsules as formed in Examples 10 to 13, microcapsules were
coated with palmitic or behenic acids. The microcapsules 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.
[0128] The efficacy of coating was assessed by an indirect method
derived from our previous ultrasound studies. The hollow nature of
the microcapsules means that an echo signal may be obtained by
reflection from the suspended air bubbles using echogenic assay
developed for our ultrasound contrast agent. Ultrasound images were
gathered from a beaker of water containing 1.times.10.sup.6
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.
[0129] The uncoated microcapsules very quickly lost all air and
thus the potential to reflect ultrasound. However, as shown in
Table 12, coated microcapsules retained their structure for a
longer period and hence showed a prolonged signal over several
minutes.
14TABLE 12 Echogenicity of Coated HSA Microcapsules Time (min) HSA
only HSA/Palmitic HSA/Behenic 0 1.750 1.911 0.878 5 0.043 0.482
0.524 10 0 0 0.004
EXAMPLE 15
[0130] Soluble mannitol microcapsules were prepared as set out in
Example 10 (15% aqueous mannitol spray-drying feedstock) and coated
with palmitic acid and behenic acid as described in Example 14. 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. The results are shown in
Table 13.
15TABLE 13 Echogenicity of Coated Mannitol Microcapsules Analysis
Time Mannitol Mannitol/Palmitic Mannitol/Behenic 0 1.632 1.732
0.924 10 min 0.325 0.502 0.235 17 hours 0.00 0.842 not
determined
EXAMPLE 16
[0131] Using samples of the microcapsules produced in the Examples
10 to 18, 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.
[0132] 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.
[0133] 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.
[0134] The aerolisation behaviour of the samples was tested by
connecting the inhaler to a vacuum chamber; 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.
EXAMPLE 17
[0135] The performance of the dry powder formulations from Examples
10 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 1 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 1 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.
EXAMPLE 18
[0136] A comparison of the dosing and deposition of fixed insoluble
microcapsules and soluble microcapsules as produced in Example 10
was made in the lung of rabbits.
[0137] 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 11 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.
[0138] 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 capsules having dissolved in the fluids of the lung.
However, the presence of FITC-lysine addudt 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.
* * * * *