Note: Descriptions are shown in the official language in which they were submitted.
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
1
PARENTERALLY ADMINISTRABLE MICROPARTICLES
TECHNICAL FIELD
The present invention lies within the field of
galenic formulations for the administration of
biologically active substances, more specifically
microparticles for controlled release primarily intended
for parenteral administration of biologically active
substances, especially drugs. More specifically, it
relates to a novel production process for such particles
containing a biologically active substance and to novel
particles for controlled release which are thereby
obtainable.
BACKGROUND TO THE INVENTION
Many drugs have to be administered by injection,
since they are either subjected to degradation or are
insufficiently absorbed when they are given, for example,
orally or nasally or by the rectal route. A drug
preparation intended for parenteral use has to meet a
number of requirements in order to be approved by the
regulatory authorities for use on humans. It must
therefore be biocompatible and biodegradable and all used
substances and 'their degradation products must be non-
toxic. In addition, particulate drugs intended for
injection have to be small enough to pass through the
injection needle, which preferably means that they should
be smaller than 200 ~,m. The drug should not be degraded
in the preparation to any great extent during production
or storage thereof or after administration and should be
released in a biologically active form with reproducible
kinetics.
One class of polymers which meets the requirements
of biocompatibility and biodegradation into harmless end
products is the linear polyesters based on lactic acid,
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
2
glycolic acid and mixtures thereof. These polymers will
also hereinafter be referred to as PLGA. PLGA is degraded
by ester hydrolysis into lactic acid and glycolic acid
and has been shown to possess excellent biocompatibility.
The innocuous nature of PLGA can be exemplified,
moreover, by the approval by the regulating authorities,
including the US Food and Drug Administration, of several
parenteral delayed release preparations based on these
polymers.
Parenterally administrable delayed release
products currently on the market and based on PLGA
include Decapeptyl T~ (Ibsen Biotech), Prostap SRTM
(Lederle), Decapeptyl~ Depot (Ferring) and Zoladex~
(Zeneca). The drugs in these preparations are all
peptides. In other words, they consist of amino acids
condensed into a polymer having a relatively low degree
of polymerization and they do not have any well-defined
three-dimensional structure. This, in turn, usually
allows the use of relatively stringent conditions during
the production of these products. For example, extrusion
and subsequent size-reduction can be utilized, which
techniques would probably' not be allowed in connection
with proteins, since these do not, generally speaking,
withstand such stringent conditions.
Consequently, there is also a need for controlled
release preparations for proteins. Proteins are similar
to peptides in that they also consist of amino acids, but
the molecules are larger and the majority of proteins are
dependent on a well-defined three-dimensional structure
as regards many of their properties, including biological
activity and immunogenicity. Their three-dimensional
structure can be destroyed relatively easily, for example
by high temperatures, surface-induced denaturation and,
in many cases, exposure :to organic solvents. A very
serious drawback connected with the use of PLGA, which is
an excellent material per se, for delayed release of
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
3
proteins is therefore the need to use organic solvents to
dissolve said PLGA, with the attendant risk that the
stability of the protein will be compromised and that
conformation changes in the protein will lead to an
immunological reaction in the patient, which can produce
both a loss of therapeutic effect, through the formation
of inhibitory antibodies, and toxic side effects. Since
it is extremely difficult to determine with certainty
whether a complex protein has retained its three-
dimensional structure in every respect, it is very
important to avoid exposing the protein to conditions
which might induce conformation changes.
Despite intense efforts aimed at modifying the
PLGA technology in order to avoid this inherent problem
of protein instability during the production process,
progress within this field has been very slow, the main
reason probably being that the three-dimensional
structures for the majority of proteins are far too
sensitive to withstand the manufacturing conditions used
and the chemically acidic environment formed with the
degradation of PLGA matrices. The scientific literature
contains a large number of descriptions of stability
problems in the manufacture of microspheres of PLGA owing
to exposure to organic solvents. As an example of the
acidic environm~rit which is formed upon the degradation
of PLGA matrices, it has recently been shown that the pH
value in a PLGA microsphere having a diameter of about 40
~,m falls to 1.5, which is fully sufficient to denature, or
otherwise damage, many therapeutically usable proteins
(Fu et al, Visual Evidence of Acidic Environment Within
Degrading Poly(lactic-co-glycolic acid) (PLGA)
Microspheres,~ Pharmaceutical Research, Vol. 17, No. 1,
2000, 100-106). Should the microspheres have a greater
diameter, the pH value can be expected to f all further
owing to the fact that the acidic degradation products
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
4
then get more difficult to diffuse away and the
autocatalytic reaction is intensified.
The technique which is currently most commonly
used to encapsulate water-soluble substances, such as
proteins and peptides, is the use of multiple emulsion
systems. The drug substance is dissolved in an aqueous or
buffer solution and subsequently mixed with an organic
solvent, immiscible with water, containing the dissolved
polymer. An emulsion is formed which has the aqueous
phase as the inner phase. Different types of emulsifiers
and vigorous mixing are often used to create this first
emulsion. This emulsion is then transferred, under
agitation, to another liquid, usually water, containing
another polymer, for example polyvinyl alcohol, which
produces a water/oil/water triple emulsion. The
microspheres are next hardened in some way. The most
common way is to utilize an organic solvent having a low
boiling point, typically dichloromethane, and to distil
off the solvent. If the organic solvent is not fully
immiscible with water, a continuous extraction procedure
can be used by adding more water to the triple emulsion.
A number of variations of this general procedure are also
described in the literature. In certain cases, the
primary emulsion is mixed with a non-aqueous phase, for
example silicone oil. Solid drug materials can also be
used instead of dissolved ones.
PLGA microspheres containing proteins are
described in WO-Al-9013780, in which the main feature is
the use of very low temperatures during the production of
the mierospheres for the purpose of preserving high
biological activity in the proteins. The activity for
encapsulated superoxide dismutation is measured, but only
on the part which has been released from the particles.
This method has been used to produce PLGA microspheres
containing human growth hormone in WO-A1-9412158, wherein
human growth hormone is dispersed in methylene chloride
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
containing PLGA, the obtained dispersion is sprayed into
a container of frozen ethanol beneath a layer of liquid
nitrogen in order to freeze the fine droplets and said
droplets are allowed to settle in the nitrogen on the
5 ethanol. The ethanol is subsequently thawed and the
microspheres start to sink in the ethanol, where the
methylene chloride is extracted in the ethanol and the
microspheres are hardened. Using this methodology, the
protein stability can be better retained than in the
majority of other processes for enclosing proteins in
PLGA microspheres, and a product has also recently been
approved by the registration authorities in the USA.
However, this still remains to be clearly demonstrated
for other proteins and the problem remains of exposing
the enclosed biologically active substance to a very low
pH during the degradation. of the PLGA matrix.
In the aforementioned methods based on
encapsulation with PLGA, the active substances are still
exposed to an organic solvent and this, generally
speaking, is harmful to the stability of a protein.
Moreover, the discussed emulsion, processes are
complicated and probably problematical in any attempt to
scale up to an industrial scale. Furthermore, many of the
organic solvents which are utilized in many of these
processes are associated with environmental problems and
their high affinity for the PLGA polymer makes their
removal difficult.
A number of attempts to solve the above-described
problems caused by exposure of the biologically active
substance to a chemically acidic environment during the
biodegradation of the microsphere matrix and organic
solvents in the manufacturing process have been
described. In order to avoid an acidic environment during
the degradation, attempts have been made to replace PLGA
as the matrix for the microspheres by a polymer which
produces chemically neutral degradation products, and in
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
6
order to avoid exposing the biologically active substance
to organic solvents, either it has been attempted to
manufacture the microspheres in advance and, only once
they have been processed and dried, to load them with the
biologically active substance, or attempts have been made
to exclude or limit the organic solvent during
manufacture of the microspheres. A process for limiting
the quantity of solvent used where polymers are used
which can only be dissolved in organic solvents is
described in WO 99/20253, in which the limitation is
obtained by the use of an aqueous PEG solution to form an
emulsion. In this publication, there is no discussion of
any technique for concentrating or solidifying the
biologically active substance to be incorporated in the
microparticles.
By way of example, highly branched starch of
relatively low molecular weight (maltodextrin, average
molecular weight about 5 000 Da) has been covalently
modified with acryl groups for the conversion of said
starch into a form which can be solidified into
microspheres and the obtained polyacryl starch has been
converted into particulate form by radical polymerization
in an emulsion with toluene/chloroform (4:1) as the outer
phase (Characterization of Polyacryl Starch
Microparticles 'as Carriers for Proteins and Drugs,
Artursson et al, J Pharm Sci, 73, 1507-1513, 1984).
Proteins were able to be entrapped in these microspheres,
but the manufacturing conditions expose the biologically
active substance to both organic solvents and high
shearing forces in the manufacture of the emulsion. The
obtained microspheres are dissolved enzymatically and the
pH can be expected to be kept neutral. The obtained
microspheres are not suitable for parenteral
administration, especially repeated administrations, for
a number of reasons. Most important of all is the
incomplete and very slow biodegradability of both the
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
7
starch matrix (Biodegradable Microspheres IV. Factors
Affecting the Distribution and Degradation of Polyacryl
Starch Microparticles, Laakso et al, J Pharm Sci 75, 962-
967, 1986) and the synthetic polymer chain which cross-
links the starch molecules. Moreover, these microspheres
are far too small, <2 ~.m in diameter, to be suitable for
injection in the tissues for sustained release, since
tissue macrophages can easily phagocytize them. Attempts
to raise the degradation rate and the degree of
degradation by introducing a potentially biodegradable
ester group in order to bond the acryl groups to the
highly branched starch failed to produce the intended
result and even these polyacryl starch microspheres were
biodegraded far too slowly and incompletely over
reasonable periods of time (BIODEGRADABLE MICROSPHERES:
Some Properties of Polyacryl Starch Microparticles
Prepared from Acrylic acid Esterified Starch, Laakso and
Sjoholm, 1987 (76), pp. 935-939, J Pharm Sci.)
Microspheres of polyacryl dextran have been
manufactured in two-phase aqueous systems (Stenekes et
al, The Preparation of Dextran Microspheres in an All
Aqueous System: Effect of the Formulation Parameters on
Particle Characteristics, Pharmaceutical Research, Vol.
15, No. 4, 1998, 557-561, and Franssen and Hennink, A
novel preparatiofi method for polymeric microparticles
without using organic solvents, Int J Pharm 168, 1-7,
1998). With this mode of procedure, the biologically
active substance is prevented from being exposed to
organic solvents but, for the rest, the microspheres
acquire properties equivalent to the properties' described
for the polyacryl starch microspheres above, which makes
them unsuitable for repeated parenteral administrations.
Bearing in mind that man does not have specific dextran-
degrading enzymes, the degradation rate should be even
lower than for polyacryl starch microspheres. The use of
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
8
dextran is also associated with a certain risk of serious
allergic reactions.
Manufacture of starch microspheres with the use of
non-chemically-modified starch using an oil as the outer
phase has been described (US 4,713,249; Schroder, U.,
Crystallized carbohydrate spheres for slow release arid
targeting, Methods Enzymol, 1985 (112),
116-128;
Schroder, U., Crystallized carbohydrate spheres as a slow
release matrix for biologically active substances, Bio-
materials 5:100-104, 1984). The microspheres are
solidified in these cases by precipitation in acetone,
which leads both to the exposure of the biologically
active substance to an organic solvent and to the non-
utilization, during the manufacturing process, of the
natural tendency of the starch to solidify through
physical cross-linking. This leads, in turn, to
microspheres having inherent instability, since the
starch, after resuspension in water and upon exposure to
body fluids, will endeavour to form such cross-links. In
order for a water-in-oil emulsion to be obtained, high
shear forces are required and the microspheres which are
formed are far too small to be suitable for parenteral
sustained release.
EP 213303 A2 describes the production of
microspheres of; inter alia, chemically unmodified starch
in two-phase aqueous systems, utilizing the natural
capacity of the starch to solidify through the formation
of physical cross-links, and the immobilization of a
substance in these microspheres for the purpose of
avoiding exposure of the biologically active substance to
organic solvents. The described methodology, in
combination with the starch quality which is defined,
does not give rise to fully biodegradable particles.
Neither are the obtained particles suitable for
injection, particularly for repeated injections over a
longer period, since the described starch quality
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
9
contains far too high quantities of foreign vegetable
protein. In contrast to what is taught by this patent, it
has now also surprisingly been found that significantly
better yield and higher loading of the biologically
active molecule can be obtained if significantly higher
concentrations of the polymers are used than is required
to form the two-phase aqueous system and that this also
leads to advantages in terms of the conditions for
obtaining stable, non-aggregated microspheres and their
size distribution. The temperature treatments which are
described cannot be used for sensitive macromolecules and
the same applies to the processing which comprises drying
with either ethanol or acetone.
Alternative methods for the manufacture of
microspheres in two-phase aqueous systems have been
described. In US 5 981 719, microparticles are made by
mixing the biologically active macromolecule with a
polymer at a pH close to the isoelectric point of the
macromolecule and stabilizing the microspheres through
the supply of energy, preferably heat. The lowest
percentage of macromolecule, i.e. the biologically active
substance, in the preparation is 40%, which for most
applications is too high and leads to great uncertainty
in the injected quantity of active substance, since the
dose of micropa~ticles becomes far too low. Even though
the manufacturing method is described as mild and capable
of retaining the biological activity of the entrapped
biologically active substance, the microparticles are
stabilized by heating and, in the examples given, heating
is effected to at least 58°C~ for 30 min. ancT, .in many
cases, to 70-90°C for an equivalent period, which cannot
be expected to be tolerated by sensitive proteins, the
biological activity of which is dependent on a three-
dimensional structure, and even where the protein has
apparently withstood the manufacturing process, there is
still a risk of small, but nonetheless not insignificant
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
changes in the conformation of the protein. As the outer
phase, a combination of two polymers is always used,
generally polyvinyl pyrrolidone and PEG, which
complicates the manufacturing process in that both these
5 substances have to be washed off from the microspheres in
a reproducible and safe way. The microparticles formed
are too small (in the examples, values below 0.1 ~,m in
diameter are quoted) to be suitable for parenteral
sustained release after, for example, subcutaneous
10 injection, since macrophages, which are cells specialized
in phagocytizing particles and which are present in the
tissues, are easily capable of phagocytizing microspheres
up to 5-10, possibly 20 Vim, and the phagocytized particles
are localized intracellularly in the lysosomes, where
both the particles and the biologically active substance
are degraded, whereupon the therapeutic effect is lost.
The very small particle size also makes the processing of
the microspheres more complicated, since desirable
methods, such as filtration, cannot be used. The
equivalent applies to US 5 849 884.
US 5 578 709 and EP 0 688 429 B1 describe the use
of two-phase aqueous systems for the manufacture of
macromolecular microparticle solutions and chemical or
thermal cross-linking of the dehydrated macromolecules to
form micropartr~les. It is entirely undesirable to
chemically cross-link the biologically active
macromolecule, either with itself or with the
microparticle matrix, since chemical. modifications of
this kind have a number of serious drawbacks, such as
reduction of the bioactivity of a sensitive protein and
risk of induction of an immune response to the new
antigenic determinants of the protein, giving rise to the
need for extensive toxicological studies to investigate
the safety of the product. Microparticles which are made
through chemical cross-linking with glutaraldehyde are
previously known and are considered generally unsuitable
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
11
for repeated administrations parenterally to humans. The
microparticles which are described in US 5 578 709 suffer
in general terms from the same drawbacks as are described
for US 5 981 719, with unsuitable manufacturing
conditions for sensitive proteins, either through their
exposure to chemical modification or to harmful
temperatures, and a microparticle size distribution which
is too narrow for parenteral, sustained release and which
complicates post-manufacture processing of the
microspheres.
WO 97/14408 describes the use of air-suspension
technology for producing microparticles for sustained
release after parenteral administration, without the
biologically active substance being exposed to organic
solvents. However, the publication provides no guidance
towards the process according to the invention or towards
the novel microparticles which can thereby be obtained.
In US 5 470 582, a microsphere consisting of PLGA
and containing a macromolecule is produced by a two-stage
process, in which the microsphere as such is first
manufactured using organic solvents and then loaded with
the macromolecule at a later stage in which the organic
solvent has already been removed. This procedure leads to
far too low a content of the biologically active
substance, generally 1-2%, and to a very large proportion
being released immediately after injection, which very
often is entirely unsuitable. This far too rapid initial
release is already very high given a 1% load and becomes
even more pronounced when the active substance content in
the microspheres is higher. Upon the degradation of the
PLGA matrix, the pH falls to levels which are generally
not acceptable for sensitive macromolecules.
That starch is, in theory, a~ very suitable,
perhaps even ideal, matrix material for microparticles
has been known for a long time, since starch does not
need to be dissolved in organic solvents' and has a
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
12
natural tendency to solidify and since there are enzymes
within the body which can break down the starch into
endogenic and neutral substances, ultimately glucose, and
since starch, presumably owing to the similarity with
endogenic glycogen, has been shown to be non-immunogenic.
Despite intense efforts, starch having properties which
enable manufacture of microparticles suitable for
parenteral use and conditions which enable manufacture of
fully biodegradable microparticles under mild conditions,
which allow sensitive, biologically active substances,
such as proteins, to become entrapped, has not been
previously described.
Starch granules naturally contain impurities, such
as starch proteins, which makes them unsuitable for
injection parenterally. In the event of unintentional
depositing of insufficiently purified starch, such as can
occur in operations where many types of operating gloves
are powdered with stabilized starch granules, very
serious secondary effects can arise. Neither are starch
granules intrinsically suitable for repeated parenteral
administrations, for the reason that they are not fully
biodegradable within acceptable time spans.
Starch microspheres made of acid-hydrolyzed and
purified starch have been used for parenteral
administration to humans. The microspheres were made by
chemical cross-linking with epichlorohydrin under
strongly alkaline conditions. The chemical modification
which was then acquired by the starch. leads to reduced
biodegradability, so that the microspheres can be fully
dissolved by endogenic enzymes, such as a-amylase, but
not converted fully into glucose as the end product.
Neither the manufacturing method nor the obtained
microspheres are suitable for the immobilization of
sensitive proteins, nor is such acid-hydrolyzed starch,
which is essentially based on hydrolyzed amylose,
suitable for producing either fully biodegradable starch
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
13
microspheres or starch microspheres containing a high
load of a biologically active substance, such as a
protein.
Hydroxyethyl starch (HES) is administered
parenterally to humans in high doses as a plasma
substitute. HES is produced by starch granules from
starch consisting broadly exclusively of highly branched
amylopectin, so-called "waxy maize", being acid
hydrolyzed in order to reduce the molecular weight
distribution and being subsequently hydroxyethylated
under alkaline conditions and acid-hydrolyzed once more
to achieve an average molecular weight of around 200,000
Da. After this, filtration, extraction with acetone and
spray-drying are carried out. The purpose of the
hydroxyethylation is to prolong.. the duration of the
effect, since non-modified amylopectin is very rapidly
degraded by a,-amylase and its residence time in the
circulation is around 10 minutes. HES is not suitable for
the production of fully biodegradable microspheres
containing a biologically active substance, since the
chemical modification leads to a considerable fall in the
speed and completeness of the biodegradation and results
in the elimination of the natural tendency of the starch
to solidify through the formation of non-covalent cross-
linkings. Moreover, highly concentrated solutions of HES
become far too viscous to be usable for the production of
microparticles. The use of HES in these high doses shows
that parenterally usable starch can be .manufactured, even
though HES is not usable . for the manufacture of
microspheres without chemical cross-linking or
precipitation with organic solvents.
WQ 99/00425 describes the use of heat-resistant
proteolytic enzymes with a broad pH-optimum to purge
starch granules of surface-associated proteins. The
obtained granules are not suitable for parenteral
administration, since they still contain the starch
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
14
proteins which are present within the granules and there
is a risk that residues of the added proteolytic enzymes
will be left in the granules. Neither are the granules
suitable for the manufacture of parenterally
administrable starch microspheres in two-phase aqueous
systems, since they have the wrong molecular weight
distribution to be able to be used in high enough
concentration, even after being dissolved, and, where
microspheres can be obtained, they are probably not fully
biodegradable.
The use of shearing to modify the molecular weight
distribution of starch, for the purpose of producing
better starch for the production of tablets, is described
in US 5,455,342 and WO 93/21008. The starch which is
obtained is not suitable for parenteral administration
owing to the high content of starch proteins, which might
be present in denatured form after the shearing, and
neither is the obtained starch suitable for producing
biodegradable starch microspheres for parenteral
administration or for use in two-phase aqueous systems
for the production of such starch microspheres. Shearing
has also been used to manufacture hydrocyethylstarch, as
is disclosed in WO 96/10042. However, for similar reasons
such hydrocyethylstarch i,s not either suitable for
parenteral administration or for the production of
microspheres as referred to above.
It is in many cases necessary or desirable to
modify a biologically active substance, for example a
drug, from soluble to solid form, for example in order to
improve its stability and/or enable effective production
of a formulation of the substance in question. For
example, in an encapsulation procedure which utilizes an
emulsifying operation, it can be necessary to use a solid
form of the biologically active substance to obtain
higher efficacy through the avoidance of transport to the
outer phase, or the interface between the outer and inner
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
phase, and in order to retain the biological activity of
the substance. For substances which tolerate harsh
manufacturing conditions, extrusion and grinding can be
used, but for sensitive biologically active substances,
5 such as proteins, it is a question in the vast majority
of cases of acquiring the solid form through chemical
complexing. A well known example of one such drug
preparation on the market is crystalline insulin complex-
bonded with zinc.
10 Thus it is well known that, for proteins and
peptides, complex-bonding with divalent metal ions,
preferably zinc, has long been utilized to convert the
biologically active substance into solid form. There are
however a large number of drawbacks with such procedures.
15 One drawback is that it is not possible to form usable
complexes of all interesting biologically active
substances and that many complexing agents which are used
in a research context are not acceptable for parenteral
administration. Another drawback is the often complicated
chemistry which, even in apparently simple cases, can
require a significant amount of effort in order to be
controlled and well-characterized. Another drawback is
that the regulatory authorities in certain countries
consider that even well known and marketed substances,
after such completing, are to be regarded as new chemical
substances, which lead to demands for extensive and very
expensive characterizations from the chemical, safety and
clinical aspects. Further drawbacks are introduced when
the active substance is to be converted into solid and
dry form, since this often involves spraying and drying
procedures which are equipment-intensive and in many
cases can be complex. Many sensitive substances do not
tolerate exposure to an air/water or air/organic-liquid
interface or to those shearing forces which are required
in order to form the spray droplets. Neither is it
unusual for problems in dispersing or resuspending the
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
16
substance converted into solid form, after it has been
dried, not to yield a usable result, for example owing to
the fact that these particles attach to one another in
such a way that they cannot be driven apart by the use of
acceptable conditions. In many of these procedures,
organic solvents are used which risk being harmful to
sensitive biologically active substances and to staff
coming into contact with the substances and have an
adverse effect upon the environment.
US 5,654,010 and US 5,664,808 describe the
production of a solid form of recombinant human growth
hormone, hGH, through complexing with zinc in order to
create an amorphous complex, which is then micronized
through an ultrasound nozzle and sprayed down in liquid
nitrogen in order to freeze the droplets. The liquid
nitrogen is then allowed to evaporate at a temperature of
-80°C and the resultant material is freeze-dried. Apart
from the fact that the procedure is complex and generally
difficult to apply, it comprises a spraying procedure in
which the biologically active substance is exposed to a
water/air surface and in which the amorphous form of the
protein which is formed is suspended in methylene
chloride. Methylene chloride is an entirely undesirable
organic solvent from a toxicological viewpoint, both for
the patients and. for the working staff.
A process for the production of parenterally
administrable microparticles and having the following
features would therefore be extremely desirable:
~ a process which makes it possible to entrap sensitive,
biologically active substances in microparticles with
retention of their biological activity;
~ a process by means of which biologically active
substances can be entrapped under conditions which do
not expose them to organic solvents, high temperatures
or high shear forces and which allows them to retain
their biological activity;
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
17
~ a process which permits high loading of a parenterally
administrable preparation with even sensitive,
biologically active substances;
a process by means of which a substantially fully
biodegradable and biocompatible preparation can be
produced, which is suitable for injecting parenterally
and upon whose degradation chemically neutral
endogenic substances are formed;
~ a process by means of which a parenterally inj ectable
preparation having a size exceeding 20 wm and,
preferably exceeding 30 Vim, is produced for the
purpose of avoiding phagocytosis of tissue macrophages
and which simplifies processing of the same during
manufacture;
~ a process for the production of microparticles
containing a biologically active substance, which
microparticles can be used as intermediate product in
the production of a preparation for controlled,
sustained or delayed release and which permit rigorous
quality control of the chemical stability and
biological activity of the entrapped biological
substance;
a process which utilizes a parenterally acceptable
starch which is suitable for the production of
substantially'- fully biodegradable starch
microparticles;
~ a process which makes it possible to concentrate or
solidify the biologically active substance to be
incorporated in a parenterally injectable preparation,
without the use of chemical complexing;
~ a process which makes it possible to concentrate or
solidify the biologically active substance to be
incorporated without the use of chemical complexing
and with retention of the biological activity of the
substance;
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
1$
~ a process which makes it possible to concentrate or
solidify the biologically active substance to be
incorporated in a parenterally administrable
preparation without exposure of the substance to
air/water or air/organic solvent interfaces;
~ a process which makes it possible to concentrate or
solidify the biologically active substance to be
incorporated in a parenterally administrable
preparation, without the use of a spraying process or
drying process;
~ a process which makes it possible to avoid a
reconstitution stage and/or resuspension stage of the
biologically active substance from dry state without
prior stabilization through incorporation in
microparticles;
~ a process which makes it possible to concentrate or
solidify the biologically active substance to be
incorporated without the introduction of further
chemical substances, using a two-phase aqueous system
for the manufacture of microparticles;
~ a substantially fully biodegradable and biocompatible
microparticulate preparation which is suitable for
injecting parenterally and upon whose degradation
chemically neutral endogenic substances are formed;
~ a microparticulate preparation containing a
biologically active substance and having a particle
size distribution which is suitable for coating by
means of air suspension technology and having
sufficient mechanical strength for this purpose;
~ a coated microparticulate, preparation containing a
biologically active substance, which preparation gives
a controlled release after parenteral administration.
DESCRIPTION OF THE INVENTION
According to a first aspect of the present
invention, there is provided a process for the production
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
19
' of microparticles. More specifically, it relates to the
production of microparticles containing a biologically
active substance and primarily intended for parenteral
administration of said substance to a mammal, especially
humans. Primarily, it is a question of the production of
microparticles intended for injection. Since the
microparticles are primarily intended for injection, it
is preferably a question of the production of particles
having a mean diameter within the range of 10-200 ~,m,
usually 20-100 ~,m and especially 20-80 Vim.
The expression "microparticles" is used in
connection with the invention as a general term for
particles of a certain size according to the art which is
known per se. One type of microparticles is therefore
constituted by microspheres, which have a substantially
spherical form, whilst the term microparticle can in
general include deviation from such a perfect spherical
form. The term microcapsule, which is known per se, also
falls within the expression microparticle according to
the prior art.
More specifically the process according to the
present invention comprises:
a) preparing an 'aqueous solution of the biologically
active substance to be incorporated in the
microparticle~;
b) mixing the.solution obtained in step a) with an aqueous
solution of polyethylene glycol (PEG) under such
conditions that the biologically active substance is
concentrated and/or solidified,
c) optionally washing the concentrated and/or 'solidified
biologically active substance obtained in step b),
d) mixing the concentrated and/or solidified biologically
active substance obtained in step b) or c) with an
aqueous starch solution,
e) mixing the composition obtained in step d) with an
aqueous solution of a polymer having the ability of
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
forming a two-phase aqueous system, so as to form an
emulsion of starch droplets which contain the
biologically active substance as the inner phase in an
outer phase of said polymer solution,
5 f) causing or allowing the starch droplets obtained in
step e) to solidify into starch microparticles,
g) drying the starch microparticles from step f), and
h) optionally applying a release-controlling shell of a
biocompatible and biodegradable polymer to the dried
10 starch microparticles from step f).
Even though it is generally possible to
incorporate biologically active substances in
microparticles in a highly effective manner, since the
biologically active substance is present in soluble form
15 during the entrapment stage, it is in certain cases
preferable for the biologically active substance to be
converted into solid form. For example, it can be a
matter of further stabilizing the biologically active
substance during the entrapment stage, of further
20 increasing the yield or the load by converting the
substance into a form which, after mixing with the inner
phase (the starch solution), cannot be distributed out in
the outer phase or to the interface between the inner and
outer phases, or of converting the substance into a form
which is as inert as possible during the manufacture of
the starch microparticles, this so that improved
properties shall be acquired, for example, in terms of
the size distribution of the microparticles.
It has thus very surprisingly been found that PEG,
which is often used as the polymer to create'the outer
phase in a two-phase aqueous system, can also be used to
concentrate and/or solidify the biologically active
substance which is to be entrapped, and that this can be
realized under mild conditions which can preserve, for
example, the three-dimensional conformation and
biological activity of a protein.
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
21
This process has a number of advantages compared
with the prior art. The basis for this is that not all
biologically active substances are able to complex-bond
chemically, for example with zinc, and not all complexing
agents are acceptable for parenteral administration. In
the first place, it is not necessary to complex-bond the
biologically active substance, preferably a protein or a
peptide, in order to obtain the
concentration/solidification. In the second place, the
use of this process often also produces better stability
during the incorporation in the microparticles, compared
with soluble protein. The fact that the process does not
comprise a spraying or drying process before the
biologically active substance is incorporated in the
microparticles also means that exposure of the
biologically active substance to high shearing fopces and
to interfaces (air/water or air/organic solvent) is
avoided. Aggregation owing to electrostatic charges,
something which is very common for small, dry particles,
is also avoided. Any problems with wetting and
resuspension of a dry powder of the biologically active
substance can also be avoided. In purely general terms,
spraying processes are also complex and poorly
controlled. Neither is it necessary to utilize process
stages such as~.~reezing and slow thawing in order to
convert the biologically active substance into dry form.
It is also a distinct advantage that no organic solvents
are used to convert the biologically active substance to
the concentrated/solidified form.
For step a) of the process according to the
invention, the aqueous solution of the biologically
active substance is prepared by means of methods which
are well known within the field and which do not need
here to be described in further detail. However,
fundamental to this is that the solution is prepared
under such mild conditions, primarily in terms 'of
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
22
temperature and agitation, that the bioactivity of the
biologically active substance is preserved. Within the
field, moreover, well-known buffer substances which are
acceptable for parenteral use are often used to control
or regulate the pH value of the solution. Where required,
substances which are well known within the field and are
acceptable for parenteral use can also be used, for
example to adjust ionic strength and osmolarity. When so
desired, the obtained solution can be sterilized by means
of, for example, sterile filtration.
Through the use of the aqueous solution of
polyethylene glycol in step b), a concentration of the
biologically active substance, for example a protein, can
be obtained. This concentration often results in the
biologically active substance precipitating out, i.e.
forming a precipitate, solid particles thereby being
formed. This can be detected, for example, by examination
with a light microscope. Since the process is often
carried out quickly, the structure of the particles is
generally amorphous. Other forms of particles, for
example crystals and supercooled glass, are also covered
by the invention, however, depending on how the process
is carried out.
The term "is concentrated" also however covers the
case in which the biologically active substance does not
precipitate, but merely forms a more or less highly
viscous solution. The term "is solidified" thus also
covers the case in which a highly viscous solution of
this kind forms such stable droplets that, in practice,
it can be handled and incorporated in micropart'icles in
substantially the same way as if it were a precipitation.
The concentrated/solidified biologically active substance
can be found in the microparticle matrix in the form of
islands or discrete particles.
One embodiment of the process according to the
invention is thus represented by the case in which step
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
23
b) is performed such that the solidification of the
biologically active substance leads to a precipitation of
the same.
In another embodiment, step b) is performed such
that the solidification of the biologically active
substance results in a highly viscous solution, which has
the ability of forming droplets which can be handled at
room temperature.
In a further embodiment of the process, step b) is
performed to form a reversibly solidified active
substance.
In yet another embodiment of the process, the
solidified biologically active substance forms a pellet
or a highly viscous or solid bottom phase in
centrifugation or ultraeentrifugation.
By "reversibly solidified" is meant, in general
terms, that the biologically active substance in
question, when dissolved. in a medium suitable for each
unique biologically active substance and under suitable
conditions, and/or when released from the microparticles
in vitro and/or in vivo, is restored to essentially the
same form, both chemically and biologically, as it had
prior to the concentration/solidification with
polyethylene glycol.
That the-solidified biologically active substance
forms a pellet or a highly viscous or solid bottom phase
in centrifugation or ultracentrifugation provides a means
of detecting the desired concentration/solidification.
This means, moreover, that the substance in question is
present in another physical form than the soluble form
which is present in step a) after the preparation of the
aqueous solution.
That the biologically active substance is present
in concentrated form means, in general terms, that it is
present in a concentration which exceeds the
concentration obtainable when the substance in question
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
24
is dissolved in an aqueous medium, with or without
stabilizers and solubility-promoting substances, and with
the retention of biological activity and chemical
stability.
A combination of molecular weight and
concentration of the PEG such that the desired
concentration and/or precipitation of the biologically
active substance is obtained should be chosen. Such
conditions can simply be tried out for each specific
biologically active substance as they will be dependent
on the properties of the biologically active substance,
for example molecular weight and solubility. The
molecular weight of the PEG can be in the range of 400-
100,000 Daltons, more preferably 4 000-35 000 Daltons,
even more preferably 6 000-20,000 Daltons, and most
preferably 20,000 Daltons. The concentration of the PEG
can be in the range of 1-50 0, preferably 2-45 %, more
preferably 10-40 % and most preferably 20-35 %. That a
concentration and/or precipation has been obtained can be
investigated as above. That the biologically active
substance has retained its bioactivity is most easily
measured at this stage by dilution, for example in a
suitable buffer solution, and chemical analysis of the
biologically active substance, or alternatively by
suitable immunol.ogical and/or animal assays. If
unsatisfactory results are obtained in the initial trials
adjustment of pH, the buffer substance, or buffer
substances used, and their concentration, temperature
and/or inclusion of stabilizers known in the art should
be investigated, as well as a change in the concentration
and mean molecular weight of the PEG used, such
adjustments being readily available to anyone skilled in
the art. This step may obviously also be performed under
an inert atmosphere to avoid oxidation reactions, the
simplest way being to purge the oxygen in the solution by
an inert gas, like nitrogen or helium. For extremely
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
sensitive substances it may be necessary to use a very
pure PEG to avoid, for example, oxidation reactions.
The extent to which step c) of the process
according to the invention needs to be executed or not,
5 i.e. whether the obtained concentrated and/or solidified
active substance should be washed and, if so, to what
extent, has to be determined in each individual case and
depends, inter alia, on the proportion of the
biologically active substance which is present in
10 dissolved form in the PEG solution, on whether the
dissolved substance is sufficiently stable in this form
to be able to be incorporated in the microparticles
without far too large a quantity of undesirable
degradation products being formed, on the effect which
15 this dissolved substance has on the manufacture of the
microparticles, on whether other conditions are required
to be used, for example in terms of the concentration and
average molecular weight of PEG, as well as pH and ionic
strength, than those employed in step b), on whether PEG
20 constitutes a stabilizer for the biologically active
substance per se or by retaining the substance in
undissolved form or preventing adsorption to surfaces.
The actual washing of the concentrated and/or
solidified active substance can be effected by means of
25 suitable techniques established within this technical
field. In the simplest form of all, centrifugation washes
can be used and in many cases filtration can also be
used. In the latter case, conditions are preferably
employed under which the concentrated and/or solidified
active substance is not allowed to dry, since this can
lead, for example, to aggregation, and the process time
is shortened by the application of pressure. Fundamental
to this, of course, is that the liquid which is used
should not dissolve the concentrated and/or solidified
active substance and that the conditions which are
suitable should be determined for each individual
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
26
biologically active substance. In many cases, conditions
can be chosen, in terms of buffer composition, additives
and temperatures, such that this requirement is met, and
necessary information can be obtained from the literature
or via simple experiments. Naturally, polymers can be
added to avoid dissolution of the concentrated and/or .
solidified active substance and, in the simplest case of
all, the same composition of the PEG solution is used as
when the concentrationjsolidification was carried out.
A starch which is especially suitable in
connection with the process according to the invention,
as well as a process for the production thereof, is
accurately described in the Swedish patent application
No. 0003616-0. Another suitable starch is disclosed in a
copending PCT application having the same filing date as
the present application and entitled STARCH. Details
relating to the starches and their production can be
obtained, in other words, from said patent applications,
the contents of which in this regard are thus herewith
included by reference in the present text.
The most important features of such a starch will,
however, be described below. In order that fully
biodegradable microparticles with high active substance
yield shall be formed in a two-phase aqueous system and
in order that the obtained starch microparticles shall
have the properties to be described below, the starch
must generally predominantly consist of highly branched
starch, which, in the natural state in the starch
granule, is referred to as amylopectin. It should also
have a molecular weight distribution which ' makes it
possible to achieve desired concentrations and gelation
rates. In the two cases referred to in the previous
paragraph said molecular weight distribution can be
accomplished by means of shearing or acid hydrolysis,
respectively.
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
27
It may be added, in this context, that the term
"biodegradable" means that the microparticles, after
parenteral administration, are dissolved in the body to
form endogenic substances, ultimately, for example,
glucose. The biodegradability can be determined or
examined through incubation with a suitable enzyme, for
example alpha-amylase, in vitro. It is in this case
appropriate to add the enzyme a number of times during
the incubation period, so as thereby to ensure that there
is active enzyme permanently present in the incubation
mixture. The biodegradability can also be examined
through parenteral injection of the microparticles, for
example subcutaneously or intramuscularly, and
histological examination of the tissue as a function of
time.
Normally, biodegradable starch microparticles
disappear from the tissue within a few weeks and often
within one week. In those cases in which the starch
microparticles are coated with a release-controlling
shell, for example by the application of a thin layer, it
is generally this shell which determines the
biodegradability rate, which then, in turn, determines
when alpha-amylase becomes available to the starch
matrix.
The biocompatibility can also be examined through
parenteral administration of the micropartieles, for
example subcutaneously or intramuscularly, and
histological evaluation of the tissue, it being important
to bear in mind that the biologically active substance,
which often is a protein, has in itself the capacity to
induce, for example, an immune response if administered
to another species. For example, a large number of
recombinantly produced human proteins can give rise to an
immune response in test animals.
The starch must further have a purity which is
acceptable for the manufacture of a parenterally
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
28
administrable preparation. It must also be able to form
sufficiently stable solutions in sufficiently high
concentration to enable the biologically active substance
to be mixed in under conditions allowing the retention of
the bioactivity of the substance, at the same time as it
must spontaneously be able to be solidified in a
controlled manner in order to achieve stable, yet at the
same time biodegradable, microparticles. High
concentration of the starch is also important to prevent
the biologically active substance from being distributed
out to an unacceptable extent to the outer phase or to
the interface between the inner and the outer phases.
A number of preferred embodiments with regard to
the nature of the starch are as follows.
The starch preferably has an amylopectin content
exceeding 85% by weight, the molecular weight of said
amylopectin being reduced so that at least 80% by weight
of the material lies within the range of 10-10 000 kDa.
In addition, the starch preferably has an amino
acid nitrogen content of less than 50 ~g per g dry weight
of starch.
The starch preferably has a purity of at most 20
fig, more preferably at most 10 ~,g, and most preferably at
most 5 fig, amino acid nitrogen per g dry weight of starch.
The molecular weight of the abovementioned
amylopectin is preferably reduced, for instance by
shearing, by acid hydrolysis or by enzymatic hydrolysis,
for example with isoamylase, such that at least 80o by
weight of the material lies within the range of 100-4 000
kDa, more preferably 200-1 000 kDa, and most 'preferably
300-600 kDa.
In addition, the starch preferably has an
amylopectin content with the reduced molecular weight in
question exceeding 95% by weight, more preferably
exceeding 98o by weight. It can also, of course, consist
of 100% by weight of such amylopectin.
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
29
According to another preferred embodiment, the
starch is of such a type that it can be dissolved in
water in a concentration exceeding 25% by weight. This
means, in general, a capacity to dissolve in water
according to a technique which is known per se, i.e.
usually dissolution at elevated temperature, for example
up to approximately 80°C.
According to a further preferred embodiment, the
starch is substantially lacking in covalently bonded
extra chemical groups of the type which are found in
hydroxyethyl starch. By this is meant, in general, that
the starch essentially only contains groups of the type
which are found in natural starch and have not been in
any way modified, such as in hydroxyethyl starch, for
example. w
Another preferred embodiment involves the starch
having an endotoxin content of less than 25 EU/g.
A further preferred embodiment involves the starch
containing less than 100 microorganisms per g, often even
less than 10 microorganisms per g.
The starch can further be defined as being
substantially purified from surface-located proteins,
lipids and endotoxins by means of washing with an aqueous
alkali solution, reduced in molecular weight by means of
shearing or acid hydrolysis and purified from internal
proteins by means of ion-exchange chromatography,
preferably anion-exchange chromatography, or gel
electrophoresis. .
As far as the purity; in all these contexts is
r
concerned, it is in general the case that expressions of
the type "essentially" or."substantially" generally mean
at least of 900, for example 950, 99% or 99.9%.
That amylopectin constitutes the main component
part of the starch used means in general terms that its
percentage is 60-100% by weight, calculated on the basis
of dry weight of starch.
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
In certain cases, it can here be favourable to use
a lesser percentage, for example 2-15% by weight, of
short-chain amylose to modify the gelation rate in step
f). The average molecular weight of the said amylose lies
5 preferably within the range of 2.5-70 kDa, especially 5-
45 kDa. Other details regarding short-chain amylose can
be obtained from US patent specification 3,881,991.
In the formation of the starch solution which is
used in step d), heating according to a technique which
10 is known per se is in general used to dissolve the
starch. An especially preferred embodiment simultaneously
involves the starch being dissolved under autoclaving, it
also preferably being sterilized. This autoclaving is
realized in aqueous solutions, for example water for
15 injection or suitable buffer.
If the biologically active substance is a
sensitive protein or another temperature-sensitive
substance, the starch solution will have to cool to an
appropriate temperature before being combined with the
20 substance in question. What temperature is appropriate is
determined firstly by the thermal stability of the
biologically active substance, but in purely general
terms a temperature of less than ca. 60°C, preferably
less than 55°C, is appropriate.
25 AccordingJto a preferred embodiment, the active
substances) is/are therefore combined with the starch
solution at a temperature of at most 60°C, more
preferably at most 55°C, and preferably within the range
of 20-45°C, especially 30-37°C:
30 For the mixing operation in step d) , furthermore,
a weight ratio of starch: biologically active substance
within the range of 3:1 to 10 000:1 is expediently used.
As has been discussed above, it is also the case
for the mixing operation that the active substance is
concentrated/solidified with the use of a PEG solution
before being mixed with the starch solution. It is
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
31
possible to add the starch solution to the biologically
active substance or vice versa. After this, a homogeneous
distribution of the concentrated/solidified active
substance in the starch solution is created by means of a
suitable technique. Such a technique is well known within
the field, examples which might be quoted being magnetic
agitation, propeller agitation or the use of one or more
static mixers..
In the production of the starch microparticles
according to the present invention, the concentration of
starch in the solution which is to be converted to solid
form and in which the biologically active substance is to
be incorporated should be at least 20% by weight to
enable the formation of starch microparticles having good
properties. Exactly what starch concentration works best
in each individual case can be titrated out in a simple
manner for each individual biologically active substance,
where the load in the microparticles is that which is
required in the individual case. In this context, it
should be noted that the biologically active substance to
be incorporated in the microparticles can affect the two-
phase system and the gelation properties of the starch,
which also means that customary preparatory trials are
conducted for the purpose of determining the optimal
conditions in the individual case. Trials generally show
that the starch concentration should advantageously be at
least 30% by weight and in certain specific cases at
least 40 o by weight. As the highest limit, 50% by weight
is usually applicable, especially at most 45o by weight.
It is not normally possible to obtain these high starch
concentrations without the use of molecular-weight-
reduced, highly branched starches.
Regarding the polymer used in step e) for the
purpose of forming a two-phase aqueous system,
information is published, within precisely this technical
field, on a large number of polymers with the capacity to
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
32
form two-phase systems with starch as the inner phase.
All such polymers must be considered to lie within the
scope of the present invention. An especially suitable
polymer in this context, however, is polyethylene glycol.
This polyethylene glycol preferably has an average
molecular weight of 5-35 kDa, more preferably 15-25 kDa
and especially about 20 kDa.
The polymer is dissolved in suitable concentration
in water or aqueous solution, which expression also
includes buffer solution, and is temperature-adjusted to
a suitable temperature. This temperature lies preferably
within the range of 4-50°C, more preferably 10-40°C and
often 10-37°C. The concentration of the polymer in the
water-based solution is expediently at least 20% by
weight and preferably at least- 30% by weight, and
expediently at most 45% by weight. An especially
preferred range is 30-40% by weight.
The mixing operation in step e) can be performed
in many different ways, for example through the use of
propeller agitation or at least one static mixer. The
mixing is normally carried out within the temperature
range of 4-50°C, preferably 20-40°C, often about 37°C. In
a batch process, the starch solution can be added to the
polymer solution or vice versa. Where static mixers or
blenders are utilized, the operation is expediently
executed by the two solutions being pumped in two
separate pipelines into a common pipeline containing the
blenders.
The emulsion can be formed using low shearing
forces, since there is no high surf ace tension. present
between the phases in water/water emulsions, in contrast
to oil/water or water/oil emulsions, and in this case it
is primarily the viscosity of .the starch solution which
has to be overcome for the droplets to achieve a certain
size distribution. In most cases, magnetic or propeller
agitation is sufficient. On a larger scale, for example
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
33
when the quantity of microparticles to be produced
exceeds 50 g, it is expedient to use so-called baffles to
obtain even more effective agitation in the container
which is used. An alternative way of forming the
water/water emulsion is to use at least one static mixer,
the starch solution expediently being pumped at regulated
speed in a pipe in which the static mixers have been
placed. The pumping can be effected with any type of
suitable pump, provided that it gives an even flow rate
under these conditions, does not expose the mixture to
unnecessarily high shearing forces and is acceptable for
the manufacture of parenteral preparations in terms of
purity and non-leakage of unwanted substances. In those
cases, too, in which static mixers are used to create the
emulsion, it is generally advantageous to have the
solidification into microparticles take place in a vessel
with suitable agitation.
A preferred embodiment of the process according to
the invention means that in step e) the polymer solution
is added to the composition in at least two stages, in
which an addition is effected after the emulsion has been
created or has begun to be created.
It is also within the scope of the present
invention, of course, to add the polymer solutions in
many stages ancl.J to change, for example, the average
molecular weight and/or concentration of the polymer
used, for example in order to increase the starch
concentration in the inner phase where this is desirable.
The mixing operation in step e) is expediently
performed, moreover, under such conditions that the
formed starch droplets acquire the size required for the
microparticles, i.e. preferably a mean diameter, in the
dry state, within the range of 10-200 Vim, more preferably
20-100 ~m and most preferably 20-80 Vim.
In connection with the solidification of the
microparticles, it is important that this should take
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
34
place under conditions which are mild for the
incorporated biologically active substance(s). In other
words, it is primarily a question of using a temperature
which is not harmful to the current substance. In this
context, it has surprisingly been shown that the criteria
for this and for the formation of stable microparticles
with suitable size distribution can more easily be met
if, during the solidification, more than one temperature
or temperature level is used. It is especially
advantageous if the solidification process in the two-
phase system is initiated at a lower temperature than the
temperature which is used in the end phase of the
solidification. A preferred embodiment means that the
solidification is initiated within the range of 1-20°C,
preferably 1-10°C, especially around 4°C, and is
terminated within the range of 20-55°C, preferably 25-
40°C, especially around 37°C.
Confirmation that the chosen conditions are
correct or appropriate can be obtained by establishing
that the starch microparticles have a desired size
distribution, are stable during the subsequent washing
and drying operations and are dissolved substantially by
fully enzymatic means in vitro and/or that the
incorporated substance has been encapsulated effectively
and has retained bioactivity. The last-mentioned is
usually examined using chromatographic methods or using
other methods established within the art, in vitro or in
vivo, after the microparticles have been enzymatically
dissolved under mild conditions, and is an important
element in ensuring a robust and reliable manufacturing
process for sensitive, biologically active substances. It
is a great advantage for the microparticles to be able to
be fully dissolved under mild conditions, since this
minimizes the risks of preparation-induced artifacts,
which are usually found when, for example, organic
solvents are required to dissolve the microparticles,
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
which is the case, for example, when these consist of a
PLGA matrix.
The formed microparticles are preferably washed in
a suitable manner in order to remove the outer phase and
5 any surplus of active substance. Such washing is
expediently effected by filtration, which is made
possible by the good mechanical stability and suitable
size distribution of the microparticles. Washing by means
of centrifugation, removal of the supernatant and
10 resuspension in the washing medium may often also be
appropriate. In each washing process, one or more
suitable washing media are used, which generally are
buffer-containing aqueous solutions. In this connection,
sifting can also be used, if required, in order to adjust
15 the size distribution of the microparticles, for example
to eliminate the content of too small microparticles and
to ensure that no microparticles above a certain size are
present in the finished product.
The microparticles can be dried in any way
20 appropriate, for example by spray-drying, freeze-drying
or vacuum-drying. Which drying method is chosen in the
individual case often depends on what is most appropriate
for the retention of the biological activity for the
enclosed biologically active substance. Process
25 considerations also enter into the picture, such as
capacity and purity aspects. Freeze-drying is often the
preferred drying method, since, correctly designed, it is
especially mild with respect to the enclosed biologically
active substance. That the.° incorporated biologically
30 active substance has retained its bioactivity can be
established by means of analysis appropriate to the
substance after the microparticle has been enzymatically
dissolved under mild conditions. Suitable enzymes for use
in connection with starch are alpha-amylase and
35 amyloglucosidase, singly or in combination, it being
important to establish, where appropriate, that they are
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
36
free from possible proteases, which can degrade proteins.
The presence of proteases can be detected with methods
known within the field and, for example, by mixing the
biologically active substance in control trials and
determining its integrity in the usual manner after
incubation with the intended enzyme mixture under the
conditions which will afterwards be used to dissolve the
microparticles. Where the preparation is found to contain
protease contamination, it can be replaced by a
preparation which offers higher purity or is purged of
proteases. This can be done using techniques known within
the ffield, for example by chromatography with a,2_
macroglobulin bonded to suitable chromatographic
material.
In order to modify the release properties of the
microparticles, a release-controlling shell made from a
biocompatible and biodegradable polymer might also be
applied, moreover. Examples of suitable polymers in this
context are found in the prior art, and polymers of
lactic acid and glycolic acid (PLGA) can especially be
mentioned. The shell in question is preferably applied
using air suspension technology. An especially suitable
technique of this kind is described in W097/14408 and
details in this regard can thus be obtained from this
publication, the content of which is included in the text
by reference. The starch microparticles which are
obtained by means of the process according to the present
invention are extremely well suited to be coated, e.g. by
the application of a thin layer, by means of the said air
suspension technology, and .the coated microparticles
obtained are especially well suited to parenteral
administration.
When the produced microparticles are used, either
they are coated with a release-controlling outer shell or
not, and the dry microparticles are suspended in a
suitable medium, specifically to permit injection. Such
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
37
media and processes in these regards are well known
within the field and will not need here to be described
in further detail. The actual injection can be given
through a suitable needle or with a needle-free injector.
It is also possible to inject the microparticles using a
dry powder injector, without prior resuspension in an
injection medium.
Apart from the advantages which have been
discussed above, the process according to the invention
has the advantage that the yield of the biologically
active substance is generally high, that it is possible
to obtain a very high active substance content in the
microparticles whilst retaining the bioactivity of the
substance, that the obtained microparticles have the
correct size distribution for - use for parenteral,
controlled (for example delayed or sustained) release,
since they are too large to be phagocytized by
macrophages and small enough to be injectable through
small needles, for example 23G-25G, and that endogenic
and neutral degradation products are formed upon
degradation of the microparticles, by which means the
active substance, for example, can be prevented from
being exposed to an excessively low pH value. Moreover,
the process itself is especially well suited to rigorous
quality control.'
The process according to the invention is
especially interesting in connection with proteins,
peptides, polypeptides, polynucleotides and
polysaccharides or, in general, other drugs or
biologically active substances which are sensitive to or
unstable in, for example, organic solvents. Recombinantly
produced proteins are a very interesting group of
biologically active substances. Generally speaking,
however, the invention is not limited to the presence of
such substances, since the inventive concept is
applicable to any biologically active substance which can
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
38
be used for parenteral administration. Apart from in
connection with sensitivity or instability problems, the
invention can thus also be of special interest in such
cases where it would otherwise be difficult to remove
solvent or where toxicological or other environmental
problems might arise.
Examples of biologically active substances of the
above-specified type are growth hormone, erythropoietin,
interferon (a, (3, y-type), vaccine, epidermal growth
hormone, Factors IV, V, VI, VII, VIII and IX, LHRH-
analogue, insulin, macrophage-colony-stimulating factor,
granulocyte-colony-stimulating factor and interleukin.
Usable biologically active substances of the non
protein drug type can be chosen from the following
groups:
Antitumour agents, antibiotics, anti-inflammatory
agents, antihistamines, sedatives, muscle-relaxants,
antiepileptic agents, antidepressants, antiallergic
agents, bronchodilators, cardiotonic agents,
antiarrhythmic agents, vasodilators, antidiabetics,
anticoagulants, haemostatic agents, narcotics and
steroids.
According to another aspect of the invention, this
also relates to novel microparticles of the type which
can be produced'by means of the process according to the
invention. The novel microparticles according to the
invention are not limited, however, to those which can be
produced by means of the said process, but comprise all
microparticles of the type in question irrespective of
the production methods.
More precisely, it is a question of microparticles
ssuitable for parenteral administration, preferably via
injection, to a mammal, especially a human being, and
containing a biologically active substance, which
microparticles essentially have the same properties as
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
39
the microparticles obtainable by means of the process
described above.
According to one aspect of the invention, these
are represented by microparticles essentially consisting
of parenterally administrable, biodegradable starch as
the matrix, which contains the biologically active
substance in essentially non-chemically complex-bonded
form and in the form of solid particles having a mean
size within the range of 0.05-30 ~.m.
By mean size is usually meant, in this context,
mean diameter, at least in the case of spherical or
substantially spherical particles. In another
configuration, reference is generally to the mean value
for the largest extent of the particle in any direction.
According to one embodiment of the invention, the
particles of the biologically active substance are
obtained by precipitation, i.e. are present in
precipitated form.
The solid particles preferably have a mean size
within the range of 0.2-10 ~,m, more preferrably 0.5-5 Vim,
and most preferably 1-4 ~,m.
Another embodiment is represented. by
microparticles in which the starch has an amino acid
nitrogen content of less than 50 ~,g per g dry weight of
starch and which inicroparticles have no covalent chemical
cross-linking between the starch molecules.
Another embodiment relates to microparticles in
which the starch has an amylopectin content exceeding 85
percent by weight, of which at~least 80 percent by weight
has a average molecular weight within the range.of 10-1
000 kDa.
The starch can otherwise have the features which
have been discussed in connection with the process.
Preferably, the microparticles also have a
release-controlling shell of the type discussed in
connection with the process. Reference is also made to
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
the process regarding preferred variants of the said
shell.
Other microparticles according to the invention
are those in which the bioactivity of the biologically
5 active substance is at least 800, preferably at least 90%
and most preferably essentially maintained compared with
the bioactivity exhibited by the substance prior to its
incorporation in the starch.
Other microparticles according to the invention
10 are those which are biodegradable in vitro in the
presence of alpha-amylase and/or amyloglucosidase.
Others still are those which are biodegradable and
are eliminated from tissue after subcutaneous or
intramuscular administration. The biologically active
15 substance is preferably a protein- and more preferably a
recombinantly produced protein.
The protein is preferably chosen from amongst
protein hormones, preferably growth hormones, coagulation
factors, preferably FVII, VII, VIII and IX, LHRH
20 analogues, insulin and insulin analogues, C-peptide,
glucagon-like peptides, LHRH analogues, leptines, colony-
stimulating factors, preferably macrophage-colony-
stimulating factor, granulocyte-stimulating factor and
granulocyte/macrophage-stimulating f actor, interferons,
25 preferably interferon a, interferon ~3 and interferon y,
interleukins, and recombinantly produced vaccines.
More preferably the protein is a growth hormone,
especially a human growth hormone (hGH)..
That the biologically: active substance, in the
r
30 starch matrix is present in essentially non-chemically
complex-bonded form means in general that the molecular
ratio of total metal cations:biologically active
substance is less than 0.2:1.
According to the prior art, it is primarily zinc
35 which has been utilized for complex-bonding in similar
context. Thus, the microparticles according to the
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
41
invention have the advantage that they are essentially or
wholly lacking in such zinc.
More preferably, the abovementioned molecular
ratio of metal cations:biologically active substance is
less than 0.1:1, especially less than 0.01:1, and most
peferably, of course, as close to 0 as possible.
Where a human growth hormone constitutes the
biologically active substance, this is preferably of the
type whose dimers content is less than 2% by weight, and
more preferably less than 1% by weight, and whose
polymers content is less than 0.2% by weight, preferably
less than 0.1o by weight.
A further preferred embodiment of the
microparticles according to the invention is constituted
by those in which the biologically active substance is
human growth hormone and for which the release kinetics
for the said hGH determined in vitro are characterized by
substantially continuous and regular release over at
least one week.
Microparticles which form a parenterally
administrable, biodegradable microparticle preparation
containing a biologically active substance which, during
the first 24 hours after injection, has an active
substance release which is less than 30% of the total
release, determiried from a concentration-time graph in
the form of the ratio between area under the curve during
the first 24 hours and total area under the curve in
question.
Preferably, the release during the first 24 hours
after the injection is less~than 200, more preferably
less than 15%, even more preferably less than 10% and
imost preferably less than 5%, of the total release.
Microparticles which produce a microparticle
preparation of the abovementioned type, which, during the
first 48 hours after injection, has an active substance
release in which the maximum concentration in plasma or
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
42
serum is less than 3000 of the maximum concentration of
the biologically active substance during any point in
time in excess of 48 hours after injection.
The said maximum concentration is preferably less
than 200% and more preferably less than 100% of the
maximum concentration in question.
Another example is a microparticle preparation of
the abovementioned type, which has a biologically active
substance release in which the bioavailability of the
said substance is at least 35% of the bioavailability
obtained when the substance in question is injected
intravenously in soluble form.
The said bioavailability is preferably at least
45%, more preferably at least 50 0, of the bioavailability
obtained when the biologically_ active substance is
injected intravenously.
A further example is a microparticle preparation
of the said type which has an active substance release
characterized in that, in the release occurring during
any continuous seven-day period, the quotient of the
highest concentration of the biologically active
substance in serum or plasma divided by the mean
concentration during the said seven-day period is less
than 5, provided that the chosen seven-day period does
not include the 'first 24 hours after injection.
The said release is preferably less than 4 times,
more preferably less than 3 times, and most preferably
less than 2 times.
Another microparticle ;preparation which can be
obtained by means of the microparticles according to the
invention has a biologically active substance release in
which the mean residence time for the substance in
question is at least 4 days.
Preferably, the said mean residence time is at
least 7 days, more preferably at least 9 days, for
example at least 11 days, or especially at least 13 days.
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
43
The features which have been specified for the
above-presented microparticle preparations can be
combined in any suitable combinations whatsoever.
The different characteristics specified for the
microparticle preparation above primarily relate to the
terms MRT, burst and bioavailability.
These can be defined as follows:
MRT
An object of preparations for controlled release is
to obtain a sustained release of the active material. One
measure which can be used to quantify the release time is
mean residence time (MRT), which is the recognized term
within pharmacokinetics.
MRT is the average time for which the molecules
introduced into the body reside within the body.
(Clinical Pharmacokinetics. Concepts and Applications.
Malcolm Rowland and Thomas N. Toner; 2nd ed., Lea&Febiger,
Philadelphia London).
The MRT value can be calculated from plasma
concentration data, using the following formula.
j t~:rl~
hll~.T w ~
j ~ ~~r~ .
a
where C is the plasma concentration and t is the time.
Burst
r
A common problem with controlled release preparations
for parenteral use is that a large part of the drug is
released during the early phase immediately following
administration in the body. Within the specialist
literature, this is termed the "burst effect". This is
generally due to the fact that the drug is located on the
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
44
surface of the formulation or that the formulation (which
can consist of microparticles) bursts. A low burst effect
is very desirable, since a high concentration of drug can
be toxic and the part which disappears rapidly in the
initial period, moreover, is poorly utilized, which means
that more drug is required to maintain a therapeutic
level of the drug during the intended treatment period.
Burst is defined as that fraction of the drug which is
absorbed during the first 24 hours of the total fraction
which is absorbed.
In mathematical terms, it can be defined using "area
under curve" calculations from plasma concentration
graphs.
2:~1t
Colt
,~tl~ SI --- ~ ~ ~ QQ°~u
ao
j E'~lE
0
Bioavailability
Bioavailability is a measure of how large a part of
the supplied drug is absorbed in active form from the
site of administration to the blood. Bioavailability is
often compared with data from intravenous supply of the
drug, in which there are therefore no absorption
barriers, and is then referred to as absolute
bioavailability.
Absolute bioavailability is defined according to the
following formula:
~ .K X11'
,~ ~ ' ,f ~ ~~~ l~
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
where AUCX is the area-under-the-curve value for the
examined formulation, AUCi" is the area-under-the-curve
value for an intravenous supply of the drug, DX is the
dose of the drug in the formulation and Div is the
5 intravenous dose.
The determination of the release profile and the
pharmacokinetic parameters is preferably realized through
animal trials. The most relevant species, owing to its
similarity to humans, is the pig. Where the biologically
10 active substance can induce, during the test, an immune
response which threatens to affect the determination of
the pharmacokinetic parameters for the biologically
active substance, inhibition of the immune response
should be used, for example by drug treatment. This is
15 known within the technical field, and details can be
obtained from the scientific literature, for example
Agerso et al, (J.Pharmacol Toxicol 41 (1999) 1-8).
Other interesting microparticles according to the
invention are those which are biodegradable in vitro in
20 the presence of alpha-amylase and/or amyloglucosidase.
Further preferred microparticles are those which
are biodegradable and are eliminated from tissue after
subcutaneous or intramuscular administration.
As regards the determination of the biological
25 activity of 'the microparticles containing active
substance, this must be carried out in a manner
appropriate to each individual biological substance.
Where the determination is effected in the form of animal
trials, a certain quantity of the biologically active
30 substance incorporated in the starch microparticles is
injected, possibly after these microparticles have been
previously enzymatically dissolved under mild conditions,
and the biological response is compared with the response
obtained after injection of a corresponding quantity of
35 the same biologically active substance in a suitable
solution. Where the evaluation is made in vitro, for
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
46
example in test tubes or in cell culture, the
biologically active substance is preferably made fully
available before the evaluation by the starch
microparticles being enzymatically dissolved under mild
conditions, after which the activity is determined and
compared with the activity for a control solution having
the same concentration of the biologically active
substance in question. In any event, the evaluation shall
include any non-specific effects of the degradation
products of the starch microparticles.
EXAMPLES
The invention will now be further illustrated by the
non-limiting illustrative embodiments below.
Example 1
Procedure for the production of highly
concentrated/precipitated hGH suitable for immobilization
with PEG.
To 343 mg hGH are added 10 mM sodium phosphate
buf fer, pH 6 . 4 , to a total volume of 2 . 5 ml . PEG with a
average molecular weight of 20,000 D is dissolved in the
same buffer to a concentration of 30%, the pH being
adjusted to about 6.4. The PEG solution (25 ml) is poured
into a beaker'~having a propeller, after which the
temperature is adjusted to 15°C and the hGH solution
(about 1.25 ml) is added under propeller agitation and
the mixture allowed to stand for 75 min. under continued
agitation. The obtained suspension is centrifuged in a
Sorvall SS34 (20 min. at 5 000 rpm). The supernatant is
carefully drawn off. The precipitated protein can be
washed once with sodium acetate, pH 6.4, containing 2 mM
zinc acetate (10 ml) and the obtained supernatant is
drawn off.
Example 2
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
47
Procedure for the immobilization of PEG-solidified hGH in
starch microspheres made from highly branched sheared
starch.
Starch microspheres are made from sheared starch
with an average molecular weight of 390 kDa. The starch
is dissolved by heating in 10 mM sodium phosphate, pH
6.4, to a concentration of 40% and the obtained starch
solution is allowed to cool to about 55°C. After this,
2.1 g of the obtained starch solution are mixed with the
whole of the batch of hGH manufactured in Example 1,
suspended in 10 mM sodium acetate buffer containing 2 mM
zinc acetate, pH 6.4, total 2.9 ml, the mixture being
agitated until a homogeneous suspension of the protein. in
the starch solution is formed. To the obtained suspension
are added 12 g of a PEG solution of 42o concentration, in
which the average molecular weight of PEG is 20 kDa. The
solidification is initiated at 4°C for 17 hours and
concluded at 37°C for 6 hours. The obtained starch
microspheres containing hGH are washed three times with
38 ml 10 mM sodium acetate buffer containing 2 mM zinc
acetate, pH 6.4, and freeze-dried. The obtained
microspheres are dissolved with a,-amylase and the
quantity of incorporated hGH is determined, for example
by means of analysis with high-pressure-liquid
chromatography. 'The fraction of dimer and polymer is also
determined, for example using high-pressure-liquid
chromatography. The yield of starch microspheres
containing hGH is generally at least, 80o and the hGH
content, expressed as dry weight, is around 15 percent by
weight. The dimer content of the protein is generally <1%
and the polymer content <0.1%, which shows that the
protein is acceptable for parenteral administration to
humans.
Example 3
CA 02429100 2003-05-15
WO 02/39985 PCT/SE01/02166
48
Procedure for coating of starch microspheres containing
PEG-concentrated hGH.
The hGH-containing starch microspheres obtained in
Example 2 are coated with a release-controlling shell
made from PLGA by means of air suspension technology
according to W097/14408 with the use of a mixture
consisting of 75o RG502H and 25% RG756 (both from
Boehringer Ingelheim). After the coating operation, the
coating is dissolved with a mixture of methylene chloride
and acetone in a ratio of 1:3 and, after these solvents
have been washed away, for example by repeated
centrifugation, the microspheres are dissolved with a-
amylase. The hGH content is determined, for example by
analysis with high-pressure-liquid chromatography. The
dimer and polymer contents of .the protein are also
determined using the same technique. The protein content
can be around 11 percent by weight. The fraction of the
protein which is present in the form of dimers is <2% and
in the form of polymers <0.1%. The release kinetics for
hGH from the coated microspheres can be determined in
vitro and are characterized by the absence of an
undesirable burst and otherwise by a continuous and
regular release with a duration of around one week. With
this process, parenterally administrable microspheres can
thus be produced so as to be suitable for controlled
release of hGH.
Example 4
Procedure for the ; production of highly
concentrated/precipitated hGH suitable for immobilization
with the use of PEG.
Precipitated hGH is produced according to Example 1,
with the change that the precipitate is washed in
histidine buffer, pH 4.9.
