Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02474988 2004-08-23
POLYMERIC MATRICES
AND THEIR USES IN PHARMACEUTICAL COMPOSITIONS
The present application has been divided out of Canadian Patent Application
Serial No. 2,168,012, Canadian national phase of PCT International Publication
WO
95/06077 filed internationally August 26, 1994.
This invention relates to pharmaceutical compositions comprising polymeric
matrices, especially those containing IL-6 for use in treating diseases
mediated by IL-1
and/or TNFa, e.g., chronic inflammatory conditions. The specific polymers of
the
invention, especially the poly(ethylene carbonate) polymers described herein,
however,
are shown to be more generally useful as matrix materials in sustained release
compositions containing pharmacologically active compounds, and in particular
to have
the novel, unexpected, and highly desirable property of undergoing
nonhydrolytic
surface erosion in vivo. Therefore, matrices comprising other drugs are also
exemplified
and provided, together with processes for preparing the polymers and to
pharmaceutical
compositions containing them. Moreover, the use of IL-6 to treat conditions
mediated by
IL-1 and/or TNFa is novel and unexpected (many such conditions were previously
believed
to be exacerbated by IL-6), thus the invention further provides a new use for
IL-6 in the
treatment of, e.g., chronic pathogen-induced inflammatory conditions,
demyelinating
diseases, and acute and hyperacute inflammatory conditions such as septic
shock.
1. Treatment of diseases mediated by IL-1 and/or TNFa
Many spontaneously occuring, chronic inflammatory conditions have an
unknown, (possibly autoimmune) etiology and are believed to be mediated by IL-
1
and/or TNFa. For example, multiple sclerosis (MS), a crippling nerve disorder
characterized by disseminated patches of demyelination in the brain and spinal
cord, has occupied the attention of research organizations for many years.
Although
the precise etiology of multiple sclerosis is not fully
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understood, it is believed to have a strong autoimmune component, as
indicated, e.g., by the
inereased incidence of certain HLA antigens in patients having the disease.
Currently
available anti-inflammatory drugs such as ACTH (adrenocorticotropic hormone)
or
corticosteroids, e.g., prednisone, appear to hasten recovery in acute attacks,
especially when
administered early in the episode, but do not affect the underlying etiology
of the disease.
Long term administration of corticosteroids or immunosuppressants carries
risks of serious
side effects. A recombinant form of IFN-% was recently shown to reduce short
term plaque
formation, but has not been shown to affect the long term progression of the
disease.
Evaluation of treatment efficacy is complicated by the fact that the natural
progression of the
disease is one of spontaneous remission and chronic relapse. In short, despite
many years of
intensive research, there is so far no generally accepted specific therapy for
this very serious
disease.
Other chronic inflammatory conditions are believed to be induced by external
agents,
e.g., pathogens. For example, Lyme disease is a serious chronic condition
initiated by
infection with the tick-born spirochete Borrelia burgdorferi. Following an
initial acute phase
characterized by skin lesions and flu-like symptoms, the disease progresses to
a chronic
phase which may be characterized by arthritis and chronic neurologic
abnormalities. The
disease is usually treated with antibiotics and nonsteroidal anti-inflammatory
agents, but an
optimal therapy, particularly for the established disease, is not yet
established.
Acute or hyperacute, uncontrolled inflammatory conditions may also be caused
by
external agents, e.g., severe burns or severe infections. For example, septic
shock, and in
particular adult respiratory distress syndrome (ARDS), is a life threatening
condition for
which no effective treatment exists at present. Onset is rapid, and mortality
generally
exceeds 50%. Septic shock usually results from severe bacterial infection and
is typically
characterized by fever often followed by hypothermia in the later stages,
fluctuating blood
pressure (hyperdynamic syndrome) followed by hypotension in the later stages,
metabolic
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acidosis, impaired mental functioning, and widespread organ dysfunction,
ultimately, in many
cases, ending in death. Most commonly, septic shock results from gram-negative
bacterial
infection (endotoxic shock), but it may also result from gram-positive
bacterial infections or
other infections. The term "septic shock" as used herein is thus to be
interpreted broadly to
mean a shock state, including ARDS, resulting from a microbial infection,
especially a
bacterial infection, most especially a gram-negative bacterial infection.
IL-6 is a known cytokine. It is known to be useful in the treatment of various
conditions, e.g., thrombocytopenia and certain cancers. It is produced by the
body usually in
response to bacterial infections and has been implicated in the mediation of
inflammation,
fever, and septic shock. It is a potent immunostimulant and indeed some
literature suggests
that IL-6 driven mechanisms cause certain autoimmune or inflammatory diseases,
including
systemic lupus erythematosis, multiple sclerosis, and rheumatoid arthritis, as
well as septic
shock.
It is thus very surprising to discover that IL-6 is useful in the treatment of
chronic
inflammatory diseases (other than glomerulonephritis), e.g., multiple
sclerosis, and in the
treatment of acute and hyperacute inflammatory conditions, e.g., septic shock.
The
mechanism of this action is unclear, but without intending to be bound by any
particular
theory, we believe that, through a feedback mechanism, IL-6 can suppress or
inhibit the
expression, release or function of other cytokines, particularly TNFa and/or
IL-I, possibly by
upregulating the release of soluble TNFoc receptor and/or IL-I receptor
antagonist, thereby
suppressing the activity and resulting autoimmune, inflammatory, or shock
conditions that are
principally mediated by these cytokines. In the case of conditions
characterized by IL-6
mediated complement-activating antigen-antibody (IgG) complexes, particularly
glomerulonephritis (which is usually caused by aggregation of such complexes
in the kidney),
however, IL-6 is shown to exacerbate the condition. Thus, we have shown that
IL-6 is
curative in animal models for MS and Lyme arthritis, for example, which are
believed to be
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driven primarily by II.-I andlor TNFa, but exacerbates the glomerulonephritis
in lupus mice,
which is believed to be directly driven by IL-6. We have also shown that IL-6
is curative by
itself in mouse models of endotoxic shock, which is likewise hypothesized to
be driven
principally by IL-I and/or TNFa.
IL-6 is therefore considered to be useful as an agent for suppressing or
inhibiting the
expression, release or function of TNFa and/or IL-I, and especially in the
treatment of
inflammatory conditions other than glomerulonephritis, and in the treatment of
septic shock.
Inflammatory conditions which may be treated using IL-6 include, for example,
arthritic
conditions, particularly pathogen-induced arthritic conditions, for example,
Lyme disease
arthritis, bacterially induced arthritis, and polioarthritis; multiple
sclerosis and other
demyelinating diseases (i.e., diseases charact.erized by demyelination in the
nerves, brain,
and/or spinal cord, including, e.g., multiple sclerosis, acute disseminated
encephalomyelitis
or postinfectuous encephalitis, optic neuromyelitis, tinnitus, diffuse
cerebral sclerosis,
Schilder's disease, adrenoleukodystrophy, tertiary Lyme disease, tropical
spastic parapoesis,
and other diseases wherein demylination, especially autoimmune-mediated
demyelination, is a
major symptom); acute severe inflammatory conditions such as bums, septic
shock,
meningitis, and pneumonia; and autoimmune diseases including polychondritis,
sclerodoma,
Wegener granulamatosis, dermatomyositis, chronic active hepatitis, myasthenia
gravis,
psoriasis, psoriatic arthritis, Steven-Johnson syndrome, idiopathic sprue,
autoimmune
inflammatory bowel disease (including e.g. ulcerative colitis and Crohn's
disease), endocrine
ophthalmopathy, Graves disease, sarcoidosis, primary billiary cirrhosis,
juvenile diabetes
(diabetes mellitus type I), uveitis (anterior and posterior),
keratoconjunctivitis sicca and
vernal keratoconjunctivitis, and interstitial lung fibrosis.
The invention thus provides:
i) A method of
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inhibiting the expression, release or function of TNFa and/or IL-I;
of treating or preventing an inflammatory condition other than
glomerulonephritis;
of treating or preventing a condition mediated by IL-I and/or TNFa;
of treating or preventing any of the conditions described above;
of treating or preventing a demyelinating disease, e.g., multiple sclerosis;
of treating or preventing an externally induced inflammatory condition;
of treating or preventing an inflammatory response to a severe acute
infection, e.g.,
septic shock, meningitis, or pneumonia;
of treating burns;
of treating or preventing a chronic pathogen-induced inflammatory condition,
e.g.,
Lyme disease;
said method comprising administering a therapeutically or prophylactically
effective amount
of IL-6, e.g., a TNFa and/or IL-I inhibiting amount of IL-6, e.g., rhlL-6,
(e.g., especially
wherein IL-6 is administered as the sole therapeutic or prophylactic agent, or
optionally
adnministered in conjunction with antimicrobial or vasoactive agents, e.g.,
optionally not in
conjunction with TNFa agonists or antagonists or with anti-TNFa antibody);
optionally in
slow release or depot form, e.g., in association with a polymeric matrix,
e.g., a poly(ethylene
carbonate) matrix as further described herein, to a subject, e.g., a mammal,
e.g., a human
being, in need of such treatment or prophylaxis;
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ii) The use of II.-6, e.g., rhII.-6, in the manufacture of a medicament for
use in the
method of (i), e.g., for treating or preventing any one of the conditions
listed under (i) above,
wherein the medicament is optionally in slow release form, e.g., optionally
further
comprising a polymeric matrix, e.g., a poly(ethylene carbonate) matrix as
further described
herein;
iii) The use of IL-6, e.g., rhIL-6, for the treatment or prevention of any of
the
conditions listed under (i) above; and
iv) A pharmaceutical composition comprising IL-6, e.g., rhIL-6, for use in the
method
of (i), e.g., for treating or preventing any of the conditions described in
(i) above, optionally
in slow release form, optionally further comprising a polymeric matrix, e.g.,
a poly(ethylene
carbonate) matrix as further described herein; for example, a sustained
release composition
(i.e., a composition which biodegrades in vivo over a period of days, weeks,
or months)
comprising IL-6 in a polymeric matrix, e.g., in the form of a microparticle or
depot, e.g.,
where the polymer exhibits nonhydrolytic surface erosion in vivo, especially
any of the drug
delivery systems described herein, for use in the treatment of any of the
above-mentioned
conditions, e.g., for the treatment of a chronic inflammatory condition.
In particular, there is provided a pharmaceutical composition comprising a
pharmacologically active compound in a polymer comprising ethylene carbonate
units, which
degrades by non-hydrolytic surface erosion.
By IL-6 is meant any compound corresponding to the known varieties of
interleukin-6
(also known as interferon beta-2 (IFN-P,, ), B-cell stimulatory factor 2 (BSF-
2), interleukin
HP-1 (HR 1), hepatocyte stimulating factor (HSF), hybridoma plasmacytoma
growth factor
(HPGF), and 26kD factor). Recombinant IL-6 is preferred, although
nonrecombinant IL-6
can also be used, e.g., as produced by IL-6 secreting cancer cell lines. II.-6
is commercially
available or may be produced by known methods, e.g., as described in
EPA 0 220 574, EPA 0 257 406, EPA 0 326 120, WO 88/00206, GB 2 063 882, or
GB 2 217 327. The IL-6 may be glycosylated, e.g., as produced by eukaryotic
cells,
e.g., CHO cells, or nonglycosylated, e.g., as produced by prokaryotic cells,
e.g., E.
coli. Recombinant human IL-6 (rhIL-6) is preferred, although IL-6 is known to
be
active cross-species, so that IL-6 derived from nonhuman sources could
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also be used and is included within the meaning of IL-6 herein. Proteins
having minor
variations in the sequence of IL-6, e.g., addition, deletion, or mutation of
1, 2, 3 or more
amino acids; fusion proteins comprising IL-6 and another protein; active
fragments of IL-6;
and/or other such variant, truncated, or mutated forms of II.-6, which have IL-
6 activity are
intended to be encompassed within the meaning of IL-6 herein.
Suitable pharmaceutical compositions comprising IL-6 together with a
phatmaceutically acceptable diluent or carrier are known. The II.-6 may be
administered
parentatly, e.g., in the form of an injectable solution or suspension, e.g.,
according to or
analogously to description in Remin on's Pharmaceutical Sciencx, 16th ed.
(Mack
Publishing Company, Easton, PA 1980). Suitable carriers include aqueous
carriers such as
saline solution, Ringer's solution, dextrose solution, and Hank's solution, as
well as
nonaqueous carriers such as fixed oils and ethyl oleate. For ordinary parental
administration,
the IL-6 is available in lyophilized form in unit dose amounts which may be
mixed with the
carrier to form a suitable solution or suspension for injection.
Alternatively, the IL-6 may be administered using an implantable or sustained
release
drug delivery system, e.g., in microparticle or depot form in association with
a polymer, to
form a polymeric matrix whereby the drug is released slowly from the matrix.
This is
preferred, e.g., where the condition to be treated is chronic, e.g., a chronic
inflatnmatory
condition, and the requisite treatement extends over a period of weeks or
months. By
polymer is meant any suitable (e.g., pharmacologically acceptable) linear,
high molecular
weight molecule formed of repeating units (including homopolymers, co-
polymers, and
heteropolymers), optionally branched or crosslinked, which may be made, e.g.,
by
polymerization of a single molecule or from the co-polymerization of more than
one
molecule (e.g., poly(ethylene carbonate) from ethylene oxide and carbon
dioxide as described
below), and optionally containing interruptions in the polymer chain with
other units.
Preferably, the polymer is linear and is composed of carbon, oxygen and
hydrogen, e.g.,
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poly-DL-lactide-co-glycolide, polyethylene glycol, or poly(ethylene
carbonate). Preferably,
the polymer exhibits non-hydrolytic surface erosion, e.g., a poly(ethylene
carbonate) as herein
further described.
The dosage will of course vary depending on the exact type of II.-6 employed,
the
host, the mode of administration, and the nature and severity of the condition
being treated.
The IL-6 is administered to larger mammals, e.g., man, by subcutaneous
injection or in
sustained release form to provide a dosage of from 0.5 g/kg/day to 30
g/kg/day, preferably
from 2.5 g/kg/day to 10 pg/kg/day, or in any other dosage which is safe and
effective for in
vivo activity in known therapeutic applications of II.-6, e.g., in a platelet-
increasing dosage.
In the case of severe acute inflammatory conditions, e.g., septic shock,
higher dosages
administered i.v. may be desirable to achieve a rapid and strong response.
Frequency of IL-6
administration may optionally be reduced from daily to every other day or
every week, or
longer in the case of sustained release forms, which are preferred when the
treatment is given
over longer periods of time. IL-6 treatment may result in chills, fever, and
flu-like
symptoms, which normally can be treated or prevented with co-adnunistration of
nonnarcotic
analgesics such as aspirin, acetaminophen or indometacin. Other significant
side effects
ordinarily appear only at higher dosages, e.g., above 10 pg/kg/day, and can
ordinarily be
relieved by reducing the dosage.
ll. Polymeric matrices for sustained release
The invention further provides pharmaceutical compositions suitable for
sustained
release of drugs, which are suitable, e.g., for administration of IL-6, e.g.
in the above
described indications, as well as for other drugs. The pharmaceutical
compositions are
especially those comprising polymers of poly(ethylene carbonate), sometimes
referred to as
as poly(ethylene carbonate)s or PECs.
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Although the prior art provides some examples of poly(ethylene carbonate)s for
use in
drug delivery systems, the prior art does not disclose the particular polymers
of the invention
and does not disclose polymers capable of undergoing nonhydrolytic surface
erosion in vivo.
The prior art also does not disclose such drug delivery systems for the
delivery of certain of
the particular drugs disclosed herein, e.g., IL-6, nor does it suggest that a
sustained release
system would be desirable for delivery of such drugs.
Particularly surprising are the degradation characteristics of the polyniers
of the
invention. On the basis of general chemical knowledge, it is expected that
carbonate ester
bonds are in principle cleavable. However, polycarbonates have been proved to
be stable
under moderate conditions in vitro.
According to Chem. Pharm. Bull. 31(4), 1400 - 1403 (1983) poly(ethylene
carbonate)s
are degradable in vivo, but the polymer tested was not clearly identified,
e.g. by modern
spectroscopical methods. According to page 1402, in vivo degradation was only
explainable
as due to the influence of hydrolytic enzynies.
According to Chem. Pharm. Bull. 32 (7), 2795-2802 (1984) microparticles were
made
of poly(ethylene carbonate) containing Dibucaine. Although the description
relates to the
firstly cited art, the release of Dibucaine was not seen to be related to in
vitro or in vivo
degradation pattern of the polymer, but to diffusion through the polymer. Also
here the
physical and chemicai properties of the poly(ethylene carbonate) tested were
not sufficiently
evaluated.
According to Makromol. Chem. 183, 2085 - 2092 (1982), especially page 2086,
carbon dioxide epoxide polymers are considered to be biodegradable and it is
said that
preliminary results confumed the biodegradability of carbon dioxide - ethylene
oxide
polymers and thus their use in controlled drug release. For support of the
allegation regarding
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.
the biodegradability Jinko Zoki 3(Suppl.), 212 (1974) was cited. In this
publication it was
said that poly(ethylene carbonate) belongs to the group of compounds which are
most easily
hydrolysed and even the enzyme pronase had no difficulty in decomposing it.
This means
that an enzymatic hydrolysis in vitro and in vivo would be possible, since
pronase is
composed of a mixture of hydrolytic enzymes. However, this commnt seems very
doubtful.
We have subjected the poly(ethylene carbonate)s of our invention in the form
of pressed
disks of 5 mm diameter and 25 mg weight to 10 mg/ml pronase and 5 mM
CaCI?.2HZ0 in
phosphate-buffered saline (PBS) of pH 7.4 and to 10 mg/ml pronase E and 5 mM
CaCI2.2HZ0 in phosphate-buffered saline of pH 7.4 (at 37 C) and no degradation
could be
observed (see Fig. 1). The pronase solution was renewed every day.
It is now surprisingly discovered that a selection of poly(ethylene
carbonate)s having a
special ethylene carbonate content, viscosity and glass transition temperature
range, which are
not degradable by hydrolysis (e.g., in the presence of hydrolytic enzymes,
e.g. pronase, or
under basic conditions) are nevertheless degradable in vitro and in vivo,
namely and
exclusively by surface erosion. The expression "surface erosion" is used in
the literature,
especially in relation to the hydrolytic degradation of polyanhydrides and
polyortho esters,
but was never clearly defined.
Surface erosion occurs, if there is a mass degradation merely at the surface
of the
polymer particles, without reduction of the molecular weight of the remaining
polymer
residue. Where in the literature it was alleged that surface erosion was
observed, molecular
weight determinations of the residual mass parallel to mass loss
determinations were never
carried out, and thus in fact surface erosion never was proved.
In fact, in almost all the hitherto tested polymers, polymer bulk erosion was
observed.
Systems exhibiting polymer bulk erosion have the significant disadvantage that
if the
polymer is loaded with a drug compound, e.g. a peptide, which is relatively
unstable under
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the influence of the biological medium to which it will be released, the drug
compound is
already contacted with the medium in the bulk part and can lose its activity
long before it is
released from the polymer. If the polymer would undergo a surface erosion,
i.e. when no
bulk erosion occurs, the embedded drug compound, e.g. the peptide, would
remain protected
from the detrimental influence of the biological medium just until the moment
that the
progressive surface erosion reaches the drug particles and the drug particle
is released from
the surface of the residual polymer mass. In case of polymer matrix drug
delivery systems
exhibiting surface erosion as opposed to bulk erosion, the drug particle is
thus exposed to the
dettimental influence of the biological medium during a shorter period of
time, thereby
allowing for longer, higher and more consistent release of pharmacalogically
active drug
from the polymer matrix.
For polyanhydrides in recent publications in Proc. Nat. Acad. Sci. USA 90, 552-
556
(1993) and 90, 4176-4180 (1993) some characteristics of a surface erosion -
like behaviour
were described. However, the whole bulk seemed to be influenced and no
molecular weight
determinations were performed. Further, this erosion is of the hydrolytic
type.
It was now discovered, that a selected group of poly(ethylene carbonate)s,
defined below,
shows, in vitro as well as in vivo, exclusively a non-hydrolytic surface
erosion.
The invention provides a polymer degradable in vivo and in vitro by surface
erosion
which is governed by a non-hydrolytic mechanism and having ethylene carbonate
units of the
formula A
-(-C(O)-O-CHZ-CHZ-O-)- A
having an ethylene carbonate content of 70 to 100 Mol k, having an intrinsic
viscosity of 0.4
to 4.0 dl/g, measured in chloroform at 20 C, and having a glass transition
temperature of
from 15 to 50 C.
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The ethylene carbonate content of the polymer according to the invention is
from 70
to 100 Mol%, particularly 80 - 100 %, preferably from 90-99.9%, such as from
94 to 99.9%.
The intrinsic viscosity of the polymer is from 0.4 to 4.0 dl/g, measured in
chloroform at
20 C. Preferably the polymer has an inherent viscosity, measured at 20 C and a
concentration
of 1 g/dl in chloroform of 0.4 to 3.0 dl/g.
Its glass transition temperature is from 15 to 50 C, preferably from 18 to
50 C.
In the literature poly(ethylene carbonate)s have been described having a glass
transition temperature of from 5 to 17 C.
The polymers of the invention are preferably made by co-polymerization of
ethylene
oxide and carbon dioxide, which production process is also a part of this
invention. As a
consequence of this production method, the polymer contains in most cases as a
co-unit the
ethylene oxide unit of the formula B
-(-CH=-CH2-O-)- B
If the polymers of the invention are exposed to an aqueous medium, e.g. a
phosphate-buffered saline of pH 7.4, practically no medium will be transponed
to their bulk
part, e.g. as is seen from Fig. 2. Therefore no bulk erosion will occur and
the remaining mass
will be kept constant (100%) for a period of at least 28 days, e.g. as shown
in the right graph
of Fig. 3.
Poly-DL-lactide-co-glycolides are at present the most commonly used matrix
materials
for sustained drug release systems. Such polymers, however, unlike the
polymers of the
invention, are degraded by hydrolysis. For example, mass degradation in PBS as
shown in
the left part of Fig. 3 for one of the most sophisticated poly-DL-lactide-co-
glycolide types,
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namely a glucose initiated poly-DL-lactide-co-glycolide (DL-PLGGLU), described
in the UK
Patent GB 2 145 422.
The difference in degradation behaviour between the poly(ethylene carbonate)s
of the
invention and the poly-DL-lactide-co-glycolides (DL-PLG) of the art in vivo is
also shown in
Fig. 3. Whereas the polylactide-co-glycolide undergoes bulk erosion, as is
seen from the
decreasing molecular weight of the residual mass of DL-PLGGLU, the molecular
weight of
the residual mass of the poly(ethylene carbonate)s remains constant (100%).
The residual mass of the total implant decreases in vivo in both cases to zero
within I
month, which means that the poly(ethylene carbonate) undergoes surface
erosion, rather than
bulk erosion. As a consequence of the absence of bulk erosion, the loaded
polymer is during
storage, i.e. before its administration, impervious to moisture and remains in
the same dry
condition in which it has been produced. Its embedded drug, if sensitive to
moisture,
remains stable.
The invention also provides a process for the production of the polymer in
which
ethylene oxide and COZ are polymerized in a molar ratio of from 1:4 to 1:5
under the
influence of a catalyst. It is clear that in the scope of this reaction the
introduction of
ethylene oxide units in the polymer chain is possible, if two epoxide
molecules react with
each other without intervention of a CO2 molecule, i.e. if an oxy anion
intermediate attacks
another ethylene oxide molecule before being carboxylated by CO2. It is thus
probable that
the polymer contains several ethylene oxide units. The polymer of the
invention, if
containing ethylene oxide units, has a random distribution of ethylene
carbonate and ethylene
oxide units according to the sum formula Am-B. _
-(C(0)-O-CH2-CH2-0-)-m (-CHZ-CH2-0-)-.
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~
in which
m x 100 = 70 to 100.
n+m
However, most of the ethylene oxide units in the polymers of the invention
have, statistically,
adjacent ethylene carbonate units, especially in those cases, in which the
molar ratio of
ethylene oxide units is small.' That means that in these cases most of the
resulting ether
functions are distributed randomly between carbonate functions along the
polymer chain.
'H-NMR spectra of the products of the invention in CDC13 confirm this
assumption.
They exhibit signals at S= ca. 4.37 ppm (Integral Ia) of the ethylene
carbonate units
(ethylene units between two carbonate functions), at ca. 4.29 and 3.73 ppm
(Integrals Ib and
Ic) of ethylene units between one carbonate and one ether function, and at ca.
3.65 ppm
(Integral Id) of ethylene units between two ether functions. The proportion of
ethylene
carbonate units (A) is then calculated within NMR accuracy limits according to
the formula:
Mol % of ethylene carbonate units (A): Ia . 100
Ia+Ib+Ic+Id
As a structural feature of poly(ethylene carbonate)s, in the literature often
their content of
ether functions, instead of their ethylene carbonate content is given. The
ratio of ether
functions (E) in the polymers of the invention may be calculated according to
the formula:
Mol % of ether functions (E) = Ic + Id . 100
Ia+lb+Ic+Id
According to the PCT-Patent Application WO 92J22600 poly(ethylene carbonate)s
are
prepared which contain ethylene oxide units and ethylene carbonate units in a
molar ratio of
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~
2 to 400 : 2, which means that the polymer contains at least 50 Mo196 of
ethylene oxide and
thus less than 50 Mol% of ethylene carbonate units. The application mentions
the
biodegradability of the polymers and their use as bioerodible matrices for the
sustained
release of phatmacologically active compounds. However, no data have been
given that the
polymers are indeed biodegradable. Generally, poly(ethylene carbonate)s having
such large
numbers of ether functions are scarcely biodegradable. The application does
not mention any
hint as to the possibility of surface erosion of the polymers.
In the Examples of the US-Patent 3 248 415 low molecular weight poly(ethylene
carbonate)s of Mw = 700 - 5000 are described having less than 70 Mol% of
ethylene
carbonate units, different from the polynwrs of the invention and nothing has
been mentioned
about their biodegradability.
According to the PCT-Application WO 89/05664 poly(ethylene carbonate)s are
described which contain in the described structure II ethylerie oxide and
ethylene carbonate
units in a molar ratio of 1 to 8: 1, which means that the polymer contains at
Ieast 50 Mo1%
of ethylene oxide and thus at most 50 Mo19b of ethylene carbonate units,
different from the
polymers of the invention. Although, the polymers are described as to be used
for
biodegradable medical devices, e.g. implants which may contain a drug
compound, no
information have been given about surface erosion.
In the process of the invention the ethylene oxide unit content and thus the
content of
ether functions, which delays or inhibits the biodegradation speed of the
polymer, is reduced
considerably by specifying the reaction conditions such as the described molar
ratio's of the
reaction components, the reaction temperature and further by choosing an
appropriate
catalyst, e.g. such prepared from Zn (C2Hs)2 and water or acetone or a di- or
a triphenol e.g.
phloroglucin, in a molar ratio of from 0.9:1 to 1:0.9 or 2:1 to 1:2
respectively, or preferably
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prepared from 7.n(CzH5)2 and a diol, especially ethylene glycol, in a molar
ratio of from 0.9:1
to 1:0.9.
The process is preferably carried out in a solvent or dispersing agent system
of an
organic solvent, e.g. dioxane and COz. COz is preferably applied in liquid
form and is present
in an excess. The pressure is preferably from 20 to 70 bar and the temperature
preferably
from 10 to 80 C, especiaUy from 20 to 70 C.
The polymers of the invention thus obtained comprise usually less than 15% of
ether
functions, preferably less than 10%, particularly less than 5%, e.g. less than
3%.
The poly(ethylene carbonate)s of the invention, if prepared using the catalyst
from ethylene
glycol or acetone and diethylzinc exhibit low polydispersities (Mw/Mn),
usually less than 5,
such as less than 2.5.
In the process according to the invention the catalyst or a part of it is
considered to be
the chain initiator for the (co)-polymer. When the reaction is finished and
the chain is
complete, its final terminal group is a hydroxyl group. The opposite site of
the chain, there
were the chain was started up, may be occupied by the catalyst group or a
fragment of it. If
the catalyst is prepared from ethylene glycol and diethylzinc or water and
diethylzine, both
ends of a polymer chain are supposed to be identical. However, if the catalyst
is prepared
from a di- or triphenol and diethylzinc, the aromatic group will be
incorporated into the end
of a chain, where the chain starts up, whereas the other end of the chain will
be a hydroxyl
group. From Fig. 4 it is seen, that poly(ethylene carbonate), if one of its
terminal groups is
blocked, e.g. by an aromatic initiator such as phloroglucin, is slower
biodegradable. For that
reason, it is assumed, that the polymer chain degradation starts at the
terminal hydroxyl
group(s). Alternatively, a later derivatization of a terminal hydroxyl group
may also be
considered, e.g. by esterification, to block terminal hydroxyl groups and to
control the
biodegradation of the poly(ethylenecarbonate)s of the invention. Suitable
terminal ester
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groups are biocompatible ester groups, like (C,-4,) fatty acid ester groups,
preferably (C,_30),
especially (C1.18) fatty acid ester groups, e.g. the ester groups of acetic
acid and stearic acid,
or a carbonic acid ester group e.g. the ethylene carbonate group, or the
pamoic acid ester
group or a lactic or glycolic or polylactic or polyglycolic or polylactic-co-
glycolic acid ester
group.
The poly(ethylene carbonate)s of the invention are stable for several hours in
hot
water (90-100 C) without considerable molecular weight reduction. A
significant increase of
the glass transition temperature is observed after exposure to boiling
bidistilled water during
hours, e.g. up to above 18 C, e.g 28 C. By performing this reaction step, a
higher polymer
purity is attained. We have found that polymers treated in this manner are
also better
processable.
The poly(ethylene carbonate) portion of the polymers of the invention is, as
said
before, not hydrolysable, that is to say during at least I month by hydrolytic
enzymes under
physiological conditions or by water at pH 12 and 37 C (see Fig. 1 and 8).
However, it was
discovered that the polymers of the invention degrade in vivo and in vitro by
surface erosion
under the influence of the superoxide radical anion 0Z'. Superoxide radical
anions 02 are
generated in inflammatory cells in vivo and ex vivo in the presence of the
poly(ethylene
carbonate)s of the invention as is seen from Fig. 5. Polylactide-co-
glycolides, the most
commonly used matrix materials for sustained drug release systems nowadays and
degraded
by bulk hydrolysis, do not induce the generation of superoxide radical anions
OZ', which is
shown in the same figure for the glucose initiated poly-DL-lactide-co-
glycolide, which was
also used for Fig. 3.
In vitro, an aqueous system was established, containing potassium superoxide
as
source of 02-- and showing surface erosion of the poly(ethylene carbonate)s of
the invention
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(see Fig. 8). In vitro a pH 12 was chosen, since the 02~ radical is sufficient
stable at this pH
value.
Interestingly, poly(propylene carbonate), different from poly(ethylene
carbonate) by
substitution of a hydrogen of the ethylene unit by a methyl group, is not at
all biodegradable,
as shown by Japanese authors in Cbem. Phaim. Bull 31(4), 1400-1403 (1983).
Using microparticle suspensions of poly(ethylene carbonate)s of the invention
a
toxicological study was conducted in 48 rats for 21 days and in 24 dogs for 35
days. Two
applications were done in each species at day 1 and day 17. After subcutaneous
and
intramuscular application of 10 and 40 mg of polymer microparticles /kg body
weight no
clinical signs of systemic toxicity, no relevant effects on hematological
parameters, on
parameters of clinical blood chemistry, on body weight, and on food
consumption were
observed. The application sites were tested for histophathological changes 4
and 21 days after
application in rats, and 18 and 35 days after application in dogs. Besides the
expected
inflammation reaction no unusual histophathological changes were found.
The degradation rate of the polymers of the invention may be adjusted within
wide
limits, depending on their molecular weight, their ethylene oxide content, the
identity of the
terminal groups, e.g. biocompatible ester groups, and the presence of 02
radical scavengers,
e.g. vitamin C. and may last between 5 days and 6 months or longer, e.g. up to
1 year. A
radical scavenger may preferably be embedded in the polymer as an additive.
The molecular weight Mw of the (co)-polymers of the invention is from 80,000,
preferably from 100,000, particularly from 200,000 to 2,000,000 Daltons,
determined by gel
permeation chromatography with methylene chloride as the eluant and
polystyrene as the
reference.
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Chem. Pharm. Bull. 32 (7) 2795-2802 (1984), discussed above, mentions that
poly(ethylene carbonate)s baving a molecular weigbt of from 50,000 to 150,000
Daltons were
used. We have found that an in vitro and in vivo degradation of the polymer
may only be
attained in a satisfactory proportion when the molecular weight is above
80,000, preferably
above 100,000 (Fig. 6); this is a preferred aspect of the invention.
The polymers of the invention may be used in phatmaceutical compositions,
especially
as matrix materials for embedding pharmacologically active compounds. Since
under in vitro
and in vivo conditions no bulk erosion occurs and the active compound is
protected by the
polymer, the active compound is released as soon as (and not before) it
appears at the matrix
surface due to surface erosion of the matrix. In an aqueous system in vitro at
pH 7.4
containing no 02', only traces of active compound were released (see Fig. 9).
A further advantage of surface erosion is that the size of the
pharmacologically active
compound molecule does not influence its release rate.
The invention therefore provides a pharmaceutical composition of a
pharmacologically
active compound in a polymer, showing non-hydrolytic surface erosion,
especially with a
linear, especially a 1:1 linear correlation of active compound release and non-
hydrolytic
polymer mass degradation and active compound protection in the polymer matrix.
The compositions are preferably used in the form of microparticles or of
implants.
The preparation of the pharmaceutical forms according to the invention may be
carried
out by methods known per se, the microparticles by appropriate spray drying or
emulsifying
techniques, the implants by mixing the drug compound and the poly(ethylene
carbonate)s
both in particulated, solid state at higher temperatures at which the
poly(ethylene carbonate)s
become soft and thus processable, optionally followed by cooling the mixture
to solid state
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and modelling it to a suitable shape. It is also possible to mix the drug
compound in
dissolved or dispersed state with a solution of the poly(ethylene carbonate)
and to evaporate
the solvent, after which the solid residue is shaped to suitable implant
forms.
Pharmaceutical compositions containing microparticles may be made by working
them
up with suitable galenical excipients and optionally bringing them in
appropriate dispensers.
Depending on the drug properties and the production process the drug loading
content
can vary between wide Iimits, in the order of 0.001 to about 70%, e.g. 0.001
to 20%,
preferably of 0.001 to 5% of weight. Percolation of medium into the polymer
due to a high
drug loading should be avoided and restricts the upper value of the loading
content.
In the medical practice of administering drug compounds every type of
pharmacologically active compound may be used in combination with the
poly(ethylene
carbonate) of the invention. In the case of microparticles preferably those
types of drug
compounds are used, which are pharmacologically active in low amounts and need
to have
an uninterrupted blood level during extended periods, e.g. hormones, peptides
or proteins,
e.g. somatostatins, interferons, or interleukins, but in particular those that
are unstable and
will desintegrate after oral use in the gastro-intestinal system and thus
preferably are
administered parenterally.
The depot formulation according to the invention may be used to administer a
wide
variety of classes of active agents, e.g. pharmacologically active agents such
as
contraceptives, sedatives, steroids, sulphonamides, vaccines, vitamines, anti-
migraine drugs,
enzymes, bronchodilators, cardiovascular drugs, analgesics, antibiotics,
antigens,
anti-convulsive drugs, anti-inflammatory drugs, anti-parkinson drugs,
prolactin secretion
inhibitors, anti-asthmatic drugs, geriatics and anti-malarial drugs. The
active agent may be
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chosen from a wide variety of chemical compounds, e.g. lipophilic and/or
hydrophilic active
agents, including peptides, such as octreotide (described in the UK patent GB
2 234 896 A).
The active proteins or peptides are preferably cytokines, e.g. interleukins, G-
CSF,
M-CSF, GM-CSF or LIF, interferons, erythropoetins, cyclosporins, or hormones,
or their
analogues, e.g. octreotide.
The pharmaceutical compositions may be used for
immunomodulation wherein the active ingredient comprisies a cytokine, e.g. an
interleukin (IL-3, II.-6), or hematopoietic colony stimulating factors (G-CSF
e.g. Filgrastim,
GM-CSF, e.g. Molgramostim, Sargramostim, M-CSF), e.g. as a vaccine adjuvant
achieving hematopoietic reconstitution after myelosuppresive therapy or after
bone
marrow transplantation, wherein the active ingredient comprises a
heinatopoetic growth
factor, e.g. GM-CSF, G-CSF, II.-3, IL-6, Leukemia Inhibitory Factor (LIF),
Stem Cell Factor
(SCF), or combinations thereof
achieving high local concentration of active ingredient, e.g., wherein the
active
ingredient comprises a drug or cytokine, GM-CSF, IL-6, IL-2, IL-4 or
combinations thereof,
to stimulate protective immune response, e.g., when administered together with
irradiated
tumor cells or vaccine antigens (an analogy to irradiated tumor cells
transfected with the
respective cytokine genes)
inducing potent immune responses wherein the active ingredient comprises,
e.g.,
GM-CSF adminstertd in combination with antigens, especially tumor antigens,
viral antigens
or bacterial antigens
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inducing wound healing with local injection of the compositions, e.g., wherein
the
active ingredient comprises GM-CSF
inducing Ag specific inunune tolerance wherein the active ingredient is, e.g.,
GM-CSF
combined with inhibitors of accessory molecules (co-receptors), especially
inhibitors for
CD28-B7 interaction, for CD40-CD40 ligand interaction, for adhesion factor
interactions
accompanying therapy with a cytostatic treatment, or as a vaccine adjuvant,
wherein
the active ingredient is e.g. a cytokine, especially an interleukin (II.-3,
Ii.-6) or cytokine
secretion inducer, e.g. a lipid derivative, e.g. the compound described in EP
0309411,
especially in Example 1, also known as MRL 953
specific immune suppression, e.g. wherein the active ingredient is an
immunophilin-
binding itnmunosuppressant, e.g., a cyclosporin (e.g., Cyclosporin A), an
ascomycin (e.g.,
FK506), or a rapamycin (e.g. rapamycin or derivative as described in WO
94/09010, e.g., 40-
O-hydroxyethyl-rapamycin)
treatment or prophylaxis of autoimmune diseases and inflammatory conditions by
slow
release of anti-inflammatory cytokines, e.g., IL-6, IL-10, or TGFO; or
interferons, e.g., IFN-
0, or Betaseron; or soluble cytokine receptors or cytokine receptor
antagonists e.g., for II.-I,
TNFa, or IL-4
treatment or prophylaxis of allergic diseases by slow release of the soluble a-
chain of
the high affinity receptor for IgE (FcE RI)
cancer treatment, e.g. with octreotide, cytokines especially interleukins,
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selective targeting e.g. for the treatment of leishmaniasis, of fungus
infections, of
enzyme storage illnesses (Tay Sachs, Gauckerillness),
AIDS or ARC therapy,
vaccination e.g. with tetanus toxoid vaccine
hematapoesis, e.g., wherein the active ingredient is erythropoetin
intraarticular injection into inflamed joints wherein the active ingredient is
an
antiinflammatory drug, especially one which are not orally bioavailable or has
a very short
half life e.g. IL-1ft converting enzynte inhibitors, metalloprotease
inhibitors.
A method for enhancing an immune response of a mammal to a vaccine comprising
administering to a mammal in need of vaccination an effective amount of GM-CSF
in
conjunction with a vaccine has been described in the international PCT-
application WO
94/01133. However, the GM-CSF was not carefully retarded in the manner
according to the
instant invention, which gives a nearly constant release of the active
compound over a longer
period of time by which the times of repeated administration of GM-CSF can be
diminished.
The invention especially provides a pharmaceutical composition of a
pharmacologically active compound in a polymer showing non-hydrolytic surface
erosion for
parenteral administration of an interleukin or CSF, particularly in a polymer
as defined
hereinbefore.
The invention also provides a method of administration such a composition to a
subject which comprises administering it parenterally to a subject in need of
such treatment.
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The depot formulations according to the invention may be used for the Icnown
indications of the particular drug compound incorporated therein.
The exact amounts of drug compound and of the depot formulation to be
administered
depends on a number of factors, e.g. the condition to be treated, the desired
duration of
treatment, the rate of release of drug compound and the degradability of the
poly(ethylene
carbonate).
The desired formulations may be produced in known manner. The amount of the
pharmacologically active agent required and the release rate thereof may be
detenmined on
the basis of known in vitro or in vivo techniques, e.g. how long a particular
active agent
concentration in the blood plasma remains at an acceptable level. The
degradability of the
matrix may also be obtained by in vitro or especially in vivo techniques, for
example
wherein the amount of matrix materials in the subcutaneous tissue is
determined after
particular time periods.
The depot formulations of the invention may be administered in the form of
e.g.
microparticles by oral, nasal or pulmonal, preferably subcutaneous,
intramuscularly or
intraveneous administration, particularly as a suspension in a suitable liquid
carrier or in the
form of implants, e.g. subcutaneously.
Repeated administration of the depot formulations of the invention may be
effected
when the polymer matrix has sufficiently been degraded, e.g. after 1, 2 or 3
weeks or I
month.
An advantage of the poly(ethylene carbonate) matrices of the invention is that
during
t-he-Telease of the-dr-ug-rompound -the -polymer chains are degraded to parts-
of a small
molecular size, which are transported by the body fluids from the site of
administration.
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Examples of drug loadings for the preferred compound octreotide are for
acromegaly,
in a parenteral liquid depot formulation having microparticles which contain
the peptide. in an
amount from at least 0.1 preferably 0.5 to 20 percent by weight relative to
the (co)-polymer
matrix, preferably 2.0 to 10, especially 3 to 6% of weight. The total dose of
octreotide is 20
to 30 mg in acromegaly and up to 100 to 200 mg in breast cancer, e.g. for 1
month of
treatment.
The release time of the peptide from the microparticles may be from 5 days to
about 2
weeks or longer.
Conveniently the sustained release formulation comprises the octreotide in the
(co)-polymer carrier, which, when administered to a rabbit or a rat
subcutaneously at a
dosage of 2 mg of octreotide per kg of animal body weight, exhibits a
concentration of
octreotide in the blood plasma of at least 0.3 ng/mi and preferably less than
20 ng/ml during
a longer period.
The pharmaceutical compositions of the invention may contain further
additives,
preferably also embedded in the (co)-polymer e.g. a radical scavenger,
especially as
scavenger of the superoxide radical anion 02.. The presence of such a
scavenger, e.g.
menadione or vitamin C, decreases the degradation rate of the poly(ethylene
carbonate) (Fig.
7).
Another type of additive is a scavenger of the hydroxyl radical, possibly
developed
under the influence of the superoxide radical anion O2', e.g. a polyol,
especially a sugar
alcohol, particularly mannitol. This additive was found to have also a
favourable influence on
body weight gain of test animals to which e.g. microencapsulated IL-3 is
administered.
Without this additive the body weigbt gain was delayed. When the compositionis
in the
form of microparticles the same additive or another may be added externally to
the existing
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microparticles, since it then has a favourable influence on the stability of a
microparticle
suspension - against flocculation and precipitation.
If an additive is present, then preferably in an amount of 1 to 90% of weight,
related
to the total weight of the formulation.
The favourable in vitro and in vivo mass degradation under the influence of
the
superoxide radical anion 02" may be seen from Fig. 8. The degradation curves
for the
residual mass are approximately linear and have a different slope, since the
degradation
conditions in vivo and in vitro are different. The amount of degraded mass per
time unit is
almost constant.
The curves for the in vivo release of a pharmacologicaUy active compound, e.g.
human II,-3, under the influence of the superoxide radical anion 02' are, like
the degradation
curves approximately linear (Fig. 10), which means that also the amount of
released drug
compound per time unit is almost constant.
A combination of both in vivo human IL-3 release and in vivo mass degradation
was
recorded in Fig. 11, showing an 1:1 correlation between in vivo mass
degradation and drug
release.
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EXAMPLES 1-5: General procedure for the synthesis of poly(ethylene
carbonate)s with a catalyst prepared from diethylzinc and water
For amount of reactants, solvent, catalyst etc. for a particular experiment
see table 1.
200 ml dry dioxane and 19.5 g (158 nunole) Zn(CTH3)2 were placed in a 750 ml
flask
under a N2-atmosphere. The flask was equipped with a mechanical sturer,
dropping funnel,
thermometer and a N2-inlet. The dropping funnel was equipped with a CaC12-
tube. The
solution was cooled down to 10 C in an ice bath and a solution of 2.7 ml of
H20 in dioxane,
see table 1) was added slowly so that the temperature was kept between 10-15
C. The
reaction mixture was stirred for additional 45 min. at room temperature, until
the initially
colorless solution turned pale yellow. This catalyst solution was transferred
to the autoclave,
treated with 40 g of CO2 and heated at 125 C for the time indicated in table
1. The mixture
was then cooled down to room temperature and 560 g (12.7 mole) CO2 was added,
followed
by slow addition of 132 g (3 mole) of ethylene oxide over a time period of 1
hour.
The reaction was allowed to proceed for the time indicated in table 1. After
this time, the
pressure was released slowly during several hours. The product, a sticky
slurry, was diluted
with dioxane and precipitated by pouring the dioxane solution into 0.25 M of
aqueous HCI.
The precipitate was dissolved in a proper amount of CHZCIZ (2-4 liter), washed
with aqueous
0:5 M HCI (2x) and with H20 (lx). The solution was dried over anhydrous Na2SO4
and
evaporated to a final volume of 0.5 to 1.5 liter, depending on the viscosity
of the solution.
The product was precipitated by pouring the CH2Cl2 solution into a 4 fold
volume of
methanol. The white precipitate was filtered off and dried ovemight at 0.5
mbar/50 C. The
crude product was reprecipitated from acetone for further purification, see
table 2. All
products provided identical 'H-NMR spectra except the relative intensities of
the signals at
3.65, 3.73, 4.29 and 4.37 ppm due to the differences in ethylene carbonate
unit content.
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TABLE 1: Experiments for the preparation of poly(ethylene carbonate)s
Example Ethylene oxide CO2 Zn(CIHs)=Dioxane Temp. Time
[mol] [mol] [mmol] [mil [ C][hl
1 3 13.6 158 300 5064
2 3 9.1 158 500 2064
3 3 13.6 158 300 20 240
4 3 13.6 158 300 2040
3 13.6 158 300 2022
6 3 13.6 238 300 5064
All experiments were run in a 1.0 litre autoclave NB2. Mol ratio H20:
Zn(C2Hs)z = 0.95 for
all experiments. The catalyst was pre-treated with 40 g of CO2 at 125 C for 1
hour except in
Example 1 (10 hours).
TABLE 2: Selected physical properties of the synthesized poly(ethylene
carbonate)s
Example Mw Mn Mw/Mn Tg inhEthylene
[kDa] [kDa] [ C] [dl/g]Carbonate
in CHC13 a) Content [9~c]
1 141.9 32.2 4.40 19.3 0.6087
2 627.3 133.5 4.70 23.5 1.4691
3 477.0 83.6 5.71 18.7 1.2791
4 758.0 97.5 7.77 20.6 1.7590
5 721.6 80.7 8.95 22.9 2.44 b)90
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6 310.9 103.1 3.02 20.1 88
a) at 20 C and a concentration of 10 mg/ml if nothing else is
indicated
b) at a concentration of I mg/mi
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EXAMPLE 7-11: General procedure for the synthesis of poly(ethylene carbonate)s
with
a catalyst prepared from diethylzinc and a diol
1. Preparation of the catalyst
200 rril of dry dioxane were placed in a dry, 4necked 750 ml flask under a
nitrogen
atmosphere. 19.50 g (158 mmol) diethylzinc were added by the mean of a glass
syringe. The
flask was equipped with a mechanical stirrer, dropping funnel, thermometer and
an argon
inlet. The dropping funnel was charged with 100 ml of dry dioxane and equipped
with a
calcium chloride tube. The apparatus was then set under an argon stream. 9.00
g (145 mmol,
0.92 molequiv.) of fresh distilled, dry ethylene glycol, (kept on molecular
sieves) were added
to the dioxane in the dropping funnel under an argon strtam. The mechanically
stirred flask
was cooled down to l0 C in an ice bath while under argon. The solution of
ethylene glycol
in dioxane was added dropwise to the stin-ed solution of diethyl zinc in
dioxane over a time
period of 30 minutes, during which time the temperature was kept between 10-14
C. An
evolution of ethane gas and precipitation was observed simultaneously on
addition of the
ethylene glycol solution. After the addition was completed, the cooling bath
was removed
and the mixture was stirred for additional 60 minutes, while allowing to warm
up to room
temperature. The heterogeneous mixture was then transferred to an autoclave (1
litre
autoclave NB2) while under argon. The autoclave was charged with ea. 40 g (0.9
mol) of
carbon dioxide and heated at 125 C for l hour under stirring to pre-treat the
catalyst with
carbon dioxide.
2. Polymerization
The autoclave with the pre-treated catalyst was cooled down to room
temperature and
was charged with additional 560 g (12.7 mol) of carbon dioxide. Then, 132 g(3
mol) of
ethylene oxide (99.8%) were added to the stirred mixture in the autoclave by
slow injection
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during I hour. After the addition was completed, the autoclave was heated to
the temperature
indicated in table 3 and the mixture was stirred for the given time at this
temperature.
3. Work-up
The autoclave was cooled down to room temperature and the pressure was
released
slowly to atomospheric pressure. The product, a white, sticky slurry, was
taken up in a total
of 7 liter of dichloromethane; 1035 ml of a 0.4 M HC1 solution were added and
the mixture
was stirred for 3 hours at room temperature. The phases were separated and the
organic layer
was washed twice with 3 liters of 0.5 M HCI and twice with 4.5 liters of
water. The
dichloromethane solution was then dried on 120 g of sodium sulfate and
concentrated to a
final volume of ca. 2 liters. The product was precipitated by slow addition of
this solution
into 6 liters of methanol. The precipiate was dried 16 hours in vacuo at 40 C
to give the
crude polymer, which was purified further as follows:
The crude product was dissolved in dichloromethane and the solution was poured
into a
fold volume of aceton during 15 minutes to precipitate the product. The
precipitate was
dried 16 hours in vacuo at 40 C to give the corresponding poly(ethylene
carbonate). The
physical properties of the products are set forth in Table 4. All products
showed strong
IR-absorptions at 1750 and 1225 cm-1. The 1 H-NMR-signal of the ethylene
carbonate units
appeared at 4.37 ppm.
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TABLE 3: Synthesis of poly(ethylene carbonate)s with a catalyst prepared from
diethylzinc
and a diol
Example Ethylene CO2[mol] Solvent'l (C.11i~)2Zn Diolbl Reaction Reaction
Oxide [ml] [mmol] [mmol] t.empera- time
[mol] ture ['C] [hrs]
7 3,0 13,6 300 158 145 20 96
8 3,0 13,6 300 158 145 50 96
9 3,0 13,6 300 158 145 60 96
3,0 13,6 300`) 158 145 50 144
11 3,0 13,6 300 158 145 ) 50 96
a) Dioxan, if nothing else indicated
b) Ethylene glycol, if nothing else indicated
c) Tetrahydrofuran as solvent instead of dioxan
d) 1,4-Butandiol instead of ethylene glycol
TABLE 4: Selected physical properties of poly(ethylene carbonate)s synthesized
using a
catalyst prepared from diethylzinc azid a diol
Example Mw Mn Mw/Mn Tg ";,.[dl/g] ethylene carbonate
[kDa] [kDa] ["C] in CHC13 content [96]
7 - - - 16.7 2.88 b) 98
8 328.0 149.0 2.20 16.4 0.97 95
9 207.0 103.0 2,00 21.2 0.65 92
10 231.0 83.8 2.76 32.6 0.72 96
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11 110.0 53.4 2.06 31.1 0.49 90
a) at 20'C and a concentration of 10 m.g/ml, if nothing else is
indicated
b) at a concentration of 1 mg/ml
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EXAMPLE 12: Experimental procedure for the synthesis of poly(ethylene
carbonate)
with a catalyst prepared from diethylzinc and phloroglncin
1. Preparation of the Catalyst
200 ml of dry dioxane were placed into a dry, 4-necked 750 ml flask under a
nitrogen
atmosphere. 19.60 g (158.7 mmol) of diethylzinc were added by the mean of a
glass syrige.
The flask was equipped with a mechanical stimr, dropping funnel, thermoawter
and an
argon inlet. The dropping funnel was charged with 100 ml of dry dioxane and
equipped with
a calcium chloride tube. The apparatus was set under an argon stream. 13.34 g
(105.8 mmol,
0.92 molequiv.) of dry phloroglucin in the dropping funnel were added to the
dioxane under
an argon stream. The mechanically stirred flask was cooled down to 10'C in an
ice bath
while under argon. The solution of phloroglucin in dioxane was added dropwise
to the stirred
solution of diethylzine in dioxane over a time period of 30 minutes, during
which time the
temperature was kept between 10-14'C. An evolution of ethane gas and
precipitation was
observed simultaneously on addition of phloroglucin solution. After the
addition was
completed, the cooling bath was removed and the mixture was stirred for
additional 30
minutes, while allowing to warm up to room temperature. The heterogeneous
mixture was
then transferred to an autoclave (I liter autoclave BN2) while under argon.
The autoclave
was charged with ca. 40 g (0.9 mol) carbon dioxide and heated at 125 C for 1
hour under
stirring to pretreat the catalyst with carbon dioxide.
2. Polymerization
The autoclave with the pre-treated catalyst was cooled down to room
temperature and
was charged with additional 560 g (12.7 mol) of carbon dioxide. Then, 132 g (3
mol) of
Ethlyene oxide (99.8%) were added to the stirred mixture in the autoclave by
slow injection
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during 1 hour. After the addition of ethylene oxide was completed, the
autoclave was stirred
at 21 C for 260 hours.
3. Work-up
The pressure of the autoclave was released slowly to atmospheric pressure. The
product
was taken up in a total of 4 liter of dichloromethane, 1035 ml of a 0.4 M HCI
solution were
added and the mixture was stirred for 3 hours at room temperature. The phases
were
separated and the organic layer was washed twice with 1.5 liters of 0.5 M HC1
and twice
with 2 liters of water. The dichloromethane solution was then dried on 120 g
of sodium
sulfate and concentrated to a final volume of ca. I liter. The product was
precipitated by
slow addition of this solution into 3 liters of methanol. The precipitate was
dried 16 hours in
vacuo at 40 C to give the crude polymer, which was purified further as
following:
The crude product was dissolved in dichloromethane and the solution was added
into a 5 fold
volume of aceton during 15 minutes to precipitate the product. The precipitate
was dissolved
again in dichloromethane, reprecipitated from methanol and dried 16 hours in
vactio at 40 C
to give the corresponding poly(ethylene carbonate).
Physical properties of the product:
Mw = 258000 Da, Mn = 35600 Da, Tg = 15.4 C.
IR: Strong absorbtions at 1751 and 1225 cm-1.
According to 1 H-NMR, the product had an ethylene carbonate content of ca.
96%.
EXAMPLE 13: Experimental procedure for the synthesis of poly(ethylene
carbonate)
with a catalyst prepared from diethyWnc and acetone
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132 g (3 Mol) of ethylene oxide were co-polymerized with 600 g (13.6 Mol) CO2
at
50 C during 96 hrs using a catalyst preparad from 8.43 g(145.16 mmol) of
acetone and
19.62 g (159 mmol) of diethylzinc.
The preparation of the catalyst as well as the polymerization were performed
similar to the
procedure described for examples 7-11, except that acetone was used instead of
a diol to
prepare the catalyst.
The poly(ethylene carbonate) thus obtained had an ethylene carbonate content
of 93%
and the following properties:
Mw = 233 kDa, Mn = 109kDa,Mw/Mn=2.14,Tg=22.4 C.
EXAMPLE 14: Synthesis of the endgroup - stearoylated poly(ethylene carbonate)
1 g of poly(ethylene carbonate) having Mw = 153000 Da, Mn = 68900 Da, Tg =
29.1
C) was dissolved in 30 ml of dry dichloromethane. The solution was treated
subsequently
with 0.98 g (12.38 mmol) of pyridine and 10 g (33.0 mmol) of stearoyl
chloride. The
reaction mixture was stirred at room temperature for 48 hours, then diluted
with 50 ml of
dichioronuthane and washed successively with 2 x 150 ml saturated sodium
bicarbonate and
water. The organic layer was dried over anhydrous sodium sulfate and the
product was
precipitated by dropwise addition of the dicbloromethane solution into 300 ml
of n-hexane.
The crude product thus obtained was purified further by dissolving in
dichloromethane and
precipitation from a 3 fold volume of diethyl ether. Finally, the product was
dried in vacuo at
40 C for 16 hours to give the endgroup - stearoylated poly(ethylene
carbonate).
Mw = 144000 Da, Mn = 71000 Da, Tg = 25.6 C.
EXAMPLE 15: Synthesis of the endgroup - acetylated poly(ethylene carbonate)
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1 g poly(ethylene carbonate) (having Mw = 153000 Da, Mn = 68900 Da, Tg =
29.1 C) was dissolved in 10 ml of dry dichloromethane. 0.98 g(12.38 mmol) of
pyridine
were added, followed by the addition of 10.08 g (98.7 mmol) of acetic
anhydride. The
reaction mixture was stuTed at room temperature for 120 hours. Then, it was
diluted with 50
ml of dichloromethane and was poured slowly onto 200 ml of saturated sodium
bicarbonate.
The mixture was stirred for 30 minutes and then the layers were separated. The
organic layer
was washed again with 150 ml of saturated sodium carbonate and finally with
water. The
dichloromethane solution was dried over anhydrous sodium sulfate and the
product was
precipitated by dropwise additional of this solution into 300 ml of diethyl
ether.
The precipitate was dissolved again in dichloromethane and reprecipitated from
diethyl ether.
The product was dried for 16 hours at 40 C in vacuo to give the poly(ethlyene
carbonate)
with the ternunal acetate ester group.
Mw = 150000 Da, Mn = 69100 Da, Tg = 26.8 C.
EXAMPLE 16: Purirication of poly(ethylene carbonate) by treatment with boiling
water
I g of poly(ethylene carbonate) (of Example 8 having Mw = 328000 Da, Mn =
149000 Da, Tg = 16.4 C) were cut into small pieces and stirred in 50 ml of
boiling bidest
water for 2 hours. The water was removed and replaced by fresh water, which
was heated
again to boiling temperature. After additional 3 hours, the polymer pieces
were isolated and
dried in vacuo at 40 C for 16 hours. The product obtained had the following
physical
properties: Mw = 340000 Da, Mn = 148000 Da, Tg = 28.3 C. Thus, a dramatic
increase of
the glass transition temperature was observed which is not attributable to a
change in the
molecular weight of the polymer.
--E7CAMPLE 17: Composition (-microparticles) with 1% hIL-3 drug loading
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1. Prenaration of drug containinQ microuarticles
1 g of poly(ethylene carbonate), Mw = 328.000 of Example 8 (PEC) was dissolved
in
ml of methylene chloride while stirring, followed by the addition of 12.1 mg
of human
interleukin 3(h1I.-3) dissolved in 0.6 ml of water. The mixture was
intensively mixed with
the Ultra-TurraxTm for one minute at 20,000 rpm (= inner W/0-phase).1 g of
gelatin A was
dissolved in 2000 ml of deionized water at 50 C and the solution was cooled
down to 20 C
(= outer W phase). The W/O-phase and the W-phase were intensively mixed.
Thereby the
inner W/O-phase was dispersed homogenously in the outer-W-phase to fine
droplets. The
resulting triple emulsion was slowly stirred for 1 hour. Hereby the methylene
chloride was
evaporated and microparticles were generated from the droplets of the inner
phase and
hardened.
After sedimentation of the microparticies the supernatant was sucked off and
the
niicroparticles were recovered by vacuum filtration or centrifugation and
rinsed with water to
eliminate gelatine. Finally, microparticles were either freeze-dried by using
mannitol as a
bulking agent or dried in a vacuum oven (mannitol free formulations) for 72
hours and
sieved (0.125 mm mesh size) to obtain the final product.
2. Placebo formulation
1 g of PEC Mw = 328.000 of Example 8 was dissolved in 10 ml of methylene
chloride while stirring (inner 0-phase). 1 g of gelatin A was dissolved in
2000 ml of
deionized water at 50 C and the solution cooled down to 20 C (=outer W phase).
The 0- and
the W-phase were intensively mixed. Thereby the 0-phase was homogenously
dispersed to
fine droplets in the outer W-phase. The resulting emulsion was slowly stinred
for 1 hour and
treated further in the manner described above.
EXAMPLES 18-26:
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All galenical formulations described hereinafter were prepared using PEC's
synthesized according to Example 8 in Table 3 and further purified in a manner
similar to
that, described in Example 16. All of them had a Mw of 300,000 to 450,000, an
ethylene
carbonate content of more than 94% and a Tg within the range of 18 to 50 C.
EXAMPLE 18: Composition (micropartides) having a 0.2% hIL-2 loading
2.9 mg of human interleukin 2 (h IL-2) was dissolved in 1.5 ml of water and IL-
2
containing nzicroparticles were prepared as described in example 17.
The microparticles were freeze-dried by using mannitol as a bullcing agent and
sieved (0.125
mm mesh size) to obtain the final product.
EXAMPLE 19: Composition (microparticles) having 0.29'c hIL-2 loading (water-
free)
The formulation was prepared as described in example 18, however, 2.9 mg of
human
Interleukin 2 were dispersed directly in the organic phase (PEC dissolved in
methylene
chloride).
EXAMPLE 20: Composition (implants) having a 0.8% hIL-3 loading
1. Compression mouldina
25 mg of microparticles, consisting of 100% (w/w) poly(ethylene carbonate)
(placebo),
99% (w/w poly(ethylene carbonate) and 1% (w/w) human interleukin-3 or 79.2%
(w/w)
poly(ethylene carbonate), 20% (w/w) mannitol and 0.8% (w/w) human interleukin-
3, were
compression moulded for 3 min at 60-70 C and 160 bar to implants (tablets) of
5 mm
diameter. The tablets were stored at 4 C in closed glass vials until use for
drug release
experiments in vitro and in vivo.
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2. Urugrelease experiments in vitro
Three tablets each of mannitol-free and mannitol-containing human interleukin-
3
formulations and placebo formulation were shaken at 37 C in synthetic culture
medium
containing I.5~'0 (v/v) N-[2-hydroxyethyl]- piperazine-N'-[2-ethanesulfonic
acid] (1 m), 10%
(v/v) fetal calf serum, and 2% (v/v) penicillin/stmptomycin solution. Samples
were drawn
from the medium at 0.5, 1, 2, 5 h and 1, 2, 3, 7, 14, 20 days and,
subsequently, the medium
was renewed. Human interleukin-3 content of the samples was measured by ELISA.
3. Drua release exveriment in vivo
Male rats, kept under optimal conditions, were anaesthesized by an inhalation
narcotic
and in each rat one tablet of the human interleukin-3 formulations and the
placebo
formulation was implanted in a subcutaneous skin pouch. After 1, 4, 7, 14, 21
days the rats
were killed by an overdose of the inhalation narcotic. The remaining tablets
were taken out,
freed from adhering tissue, and dried. Mass loss of the tablets was determined
gravimetrically. Subsequently, the human interleukin-3 content of the
remaining tablets was
measured by HPLC and ELISA.
ExamRIe 21:
Composition (w/o/w microvarticies) havine a 0.0002% - 2% hIL-2loading
4 g PEC were dissolved in 80 ml of methylene chloride with magnetic stirring.
To
this solution an appropriate amount of II.-2 (113.2 mg for 2%, 11.32 mg for
0.2% etc.)
dissolved in 6 ml of distilled water or water with some drops of ethanol was
added. The
mixture was intensively mixed with an Ultra-Turrax to disperse the IL-2
solution in the
polymer phase (= inner W/O phase). 1 g of gelatin A was dissolved in 200 ml of
1/15 M
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0
phosphate buffer (pH 7.4) at 50 C and the solution cooled down to 20'C (=
outer W phase).
The W/O- and the W-phase were intensively mixed. Thereby the inner W/O-phase
was
separated into small droplets which were dispersed homogenously in the outer W-
phase. The
resulting triple emulsion was slowly stirred for 1 hr. Hereby the aethylene
chloride was
i
evaporated and the microparticles were hardened from the droplets of the inner
phase.
After sedinientation (or centrifugation) of the microparticles the supernatant
was
sucked off and the microparticles were recovered by vacuum filtration and
rinsed with water
to eliminate gelatin. Finally, microparticles were dried in a vacuum oven for
24 hr and sieved
to obtain the final product.
The encapsulation efficiency, tested with HPLC and bioassay, was between 10
and
100%.
Example 22:
Comaosition (s/o/w microoarticies) havin¾ a 0.0002% - 2% of IL-2 )oading
The formulations were prepared as desribed in Example 21, except that IL-2 was
not
dissolved in water. Instead of dissolving IL-2, the drug was dispersed
directly into the
polymer phase (= 0-phase). The encapsulation efficiency, tested with HPLC and
bioassay,
was between 10 and 100%.
Note: The amount of polymer, methylene chloride, water and drug are varied in
a broad
range without changing the product quality. Higher drug loadings up to 20% are
obtained. In
the outer phase the gelatin is replaced by other emulsifiers such as
polyvinylalcohol etc.,
and/or the concentration of theemulsifierlbuffer are changed. Separation and
drying
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procedures described are replaced by other well known pharmaceutical
techniques such as
filtration, lyophilization and spray drying.
Exanmle 23:
Comaosition (w/o/w and s/o/w micronarticles) havina an I96 hGM-CSF load~i
The preparation was carried out according to the process described in Examples
21
and 22. As described there S/O/W and W/O/W-preparations are prepamd. However,
the
encapsulation efficiency of W/O/W formlulations was 60%, whereas S/O!W
formulations
showed lower encapsulation efficiencies.
Examules 24:
Comaosition (w/o/w and s/o(w microaarticles) havina an 1 to 10% Octreotide-
oamoate
(SMS-PA) loadina
The preparation was carried out according to the method described in Examples
19
and 20. However, SMS-PA is not water soluble. Thus, the drug was dispersed,
not dissolved,
in water, for W/O/W formulations. The encapsulation efficiency was determined
by HPLC
and was between 20 and 10096.
Example 25:
Composition (w/o/w and s/o/w microaarticies) having an I to 10% Octreotide-
acetate
loadin
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The preparation was carried out according to the method described in Examples
21
and 22. The encapsulation efficiency was determined by HPLC and was between 2
and 40%,
which is clearly lower than for the lipophilic SMS-PA.
Higher values were obtained in S/O/W formulations after using lyophilized
active compound
material (smaller drug particle).
Example 26: Octreotide aamoate (SMS-PA) release from microoarticles in rabbits
and
implants in rabbits and rats
Subcutaneous implantation of poly(ethylene carbonate) disks or injection of
poly(ethylene carbonate) microparticles (drug loading 1.95%) in an amount of
about 2 mg of
drug substance/kg body weight were performed in male rabbits (chinchilla
bastard, body
weight about 3 kg) and subcutaneous implantation of disks in male rats
(Wistar, body weight
about 375 g). Per rat and rabbit amounts of about 40 resp. 300 mg of drug
containing
polymer in the form of microparticles resp. pressed to an implant or as
suspension were
administered.
The implants disks for rats and rabbits had a diameter of 0.5 and 1 cm resp.
and were
produced as described in example 20.
To determine the drug release, blood samples were collected for 14 and 21 days
in
rats and rabbits resp. and drug residues were measured in implants by
radioimmunoassay and
HPLC.
A linear correlation of mass loss of poly(ethylene carbonate) and release of
SMS-PA
could be found (Fig. 13) as shown for high molecular mass hIL-3 (Fig. 11). A
maximum of
75% of implanted -rnaterial was degraded in 3 weeks after administration in
rabbits, a
maximum of 95% of implanted material was degraded in 2 weeks after
administration in rats.
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=
An inflammation reaction (including invasion of polymorphonuclear leucocytes
and other
cells) is a prerequisite for biodegradation of poly(ethylene carbonate). The
course of an
inflammation reaction can be expected to be species-specific giving rise to
species-specific
plasma level profiles of a drug. This was found for SMS-PA (Fig. 12). In rats,
biodegradation of poly(ethylene carbonate) is much faster than in rabbits. In
rabbits, plasma
levels of SMS-PA increase slowly to reach the phase of constant release at
about day 9
lasting until at least day 21.
Examnle 27:
Comtwsition (w/o/w microaarticles) havine a 0.0002% - 2% rhIL-6 ioadin¾
4 g PEC are dissolved in 80 ml of methylene chloride with magnetic stirring.
To this
solution an appropriate amount of rhIL-6 (113.2 mg for 2%, 11.32 mg for 0.2%
etc.)
dissolved in 6 ml of distilled water or water with some drops of ethanol is
added. The
mixture is intensively mixed with an Ultra-Turax to disperse the IL-6 solution
in the polymer
phase (= inner W/O phase). 1 g of gelatin A is dissolved in 200 ml of 1/15 M
phosphate
buffer (pH 7.4) at 50 C and the solution cooled down to 20 C (= outer W
phase). The
W/O- and the W-phase are intensively mixed. Thereby the inner W/O-phase is
separated into
small droplets which were dispersed homogenously in the outer W-phase. The
resulting triple
emulsion is slowly stirred for 1 hr. , the methylene chloride is evaporated,
and the
rnicroparticies are hardened from the droplets of the inner phase.
After sedimentation (or centrifugation) of the microparticles the supernatant
is sucked
off and the microparticles are recovered by vacuum filtration and rinsed with
water to
eliminate gelatin. Finally, microparticles are dried in a vacuum oven for 24
hr and sieved to
obtain the final-pr-oduct.
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The encapsulation efficiency, tested with HPLC and bioassay, is between 10 and
100%.
Examvle 28:
Comuosition (s/o/w microoarticles) havtg a 0.0002% - 2% of rhII,-6 loadina
The formulations are prepared as described in Example 27, except that IL-6 is
not
dissolved in water. Instead of dissolving II.-6, the drug is dispersed
directly into the polymer
phase (= 0-phase). The encapsulation efficiency, tested with HPLC and
bioassay, is between
and 100%.
Note: The amount of polymer, methylene chloride, water and drug are varied in
a broad
range without changing the product quality. Higher drug loadings up to 20% are
obtained. In
the outer phase the gelatin is replaced by other emulsifiers such as
polyvinylalcohol etc.,
and/or the concentration of the emulsifier/buffer are changed. Separation and
drying
procedures described are replaced by other well known pharmaceutical
techniques such as
filtration, lyophilization and spray drying.
Examples 29-31: Use of IL-6 in treating conditions mediated by TNFa/and or II.-
1
Example 29:
Animal model for multiple sclerosis: Chronic relansina experimentally induced
allergic
enceQhalomyelitis model in the Lewis rat (CR-EAE).
Experimentally induced allergic encephalomyelitis (EAE) in the rat is a well
studied
experimental model for multiple sclerosis in humans. [Paterson, ADV_ IMMUNOL.
5(1966)
131-208; Levine et al., AM. J. PATH. 47 (1965) 61; McFarlin et al, J. IMMUNOL.
113
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.
(1974) 712; Borel, TRANSPLANT & CLIN. DMUNOL. 13 (1981) 3]. Rats are injected
with nerve tissue from another species together with an adjuvant, and the
resulting allergic
response leads to lesions on the rat nerves which mimic the autoimmune lesions
produced in
multiple sclerosis. The rats become partially or completely paralyzed, and the
severity of the
disease is measured with and without administration of the test drugs. A
number of drugs,
such as steroids and immunosuppressants, are active in slowing the onset of
the disease but
are not capable of preventing relapses once the disease is established.
The chronic relapsing experimentally induced allergic encephalomyelitis model
(CR-
EAE) [Feiirer, et al., J. NEUROl1VIMUNOL: 10 (1985) 159-166] is therefore
considered a
particularly demanding model which closely mimics actual difficulties in
treating multiple
sclerosis patients who have established disease. In this model, the disease is
induced by
injection of a mixture of guinea pig spinal cord and Freund's complete
adjuvant enriched
with Mycobacterium tuberculosis. Typically 75 - 80 % of the sensitized rats
develop a
CR-EAE showing 2 - 3 clinical relapses during the first 40 days. After 60 - 80
days,
approximately 50 % of the rats with CR-EAE have a further relapse which is
followed by
complete recovery in only 35 % of all cases. The remaining 65 % of these
animals show a
progressive state of the disease. Drug treatment starts on day 16, after
recovery from the
first disease bout.
Recombinant human interleukin 6 (rh IL-6, Sandoz) dissolved in saline was
injected
i.p. every 2nd day starting on day 16 using 10 micrograms of IL-6 per rat (ca.
50 pg/kg).
Control animals and animals in the IL-6 group had the usual severe disease
bout (acute) at
days 11-14. On a scale of severity from 0= no disease to 4 = complete
paralysis of the
animal, the control group averaged 3.0 and the IL-6 group averaged 3.2.
Application of IL-6
every second day from day 16 to day 30 (7 applications total) resulted in an
almost complete
--inhibition of the .rlisease. Only one out_of 5_IL-6 treated rats showed.
aslight-.second disease
bout (severity 0.4). Five out of 5 control animals had a second disease bout
with a mean
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severity rating of 1.8 after day 16, and a third disease attack on days 22 -
29. No other
relapses were observed in the II.-6 treated group.
Example 30:
Animal model for arthritis: Borrelia-induced arthritis in severe combined
immunodeficiency
(SCID) mice.
Lyme arthritis (or Lyme disease arthritis) represents a unique form of chronic
arthritis
because the initiating event is known with certainty. The disease is one of
the prominent
features induced by infection with the tick-born spirochete Borrelia
bur¾dorferi. The
characteristics of synovial lesions in patients with Lynu arthritis resemble
closely those in
the synovium of patients with rheumatoid arthritis. In both patient groups
synovial lining cell
hyperthrophy, synovial cell hyperplasia, vascular proliferation and
infiltration of mononuclear
cells in the subsynovial lining ateas can be observed. Many plasma cells, high
endothelial
venules, scattered macrophages and few dendritic cells are found with intense
MHC class II
antigen presentation. In addition, cytokines, such as IL-1, IL-6 and TNF-alpha
have been
detected in synovial fluid from patients with various arthritides, suggesting
that these
cytokines may contribute to the pathogenesis of joint destruction. Recently, a
mouse model
for Lyme arthritis has been developed in SCID mi.ce which lack functional T
and B cells (M.
M. Simon, et al. (1991) Immunology Today 12: 11). The infection of the
inununodeficient
mice with Borrelia bur d~ orferi leads to a prominent and persistant
oligoarthritis. The
Borrelia-induced arthritis in SCID mice responds to corticosteroids
(prednisolon 30 mg/kg sc)
but not to immunosuppressive agents like SIM (cyclosporin A) up to doses of 30
mg/kg s.c.
It is considered a good model for cytokine-driven arthritis, including other
types of arthritis
for which the initiating event is not known with certainty.
Six week old C.B- 17 SCID mice (homozygous _for. the SCID mutationsobtained
from
Bomholtgard, Denmark, 5-6 animals/group) were inoculated with 100 mio.
Bornelia
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bur¾dorferi organisms by s.c. tailbase injection. Immunocompeteat C.B-17 mice
(same
source) were used as control animals. They do not develop any disease upon
injection of
Bonrelia bur¾dorferi . Recombinant human IL-6 (rhlL-6, Sandoz, stock sol. 5
mg/mI) was
diluted with physiological saline and was given 5 times per week for a total
of 17 injections
at a dose of 10 microgram/mouse i.p. Mice were monitored daily in blinded
fashion for
clinical signs of arthritis in the tibiotarsal and ulnacarpal joints. Clinical
arthritis was scored
according to the following parameters:
- no signs
? signs questionable
(+) reddening of joints
+ slight swelling
++ moderate swelling
+++ severe swelling of the tibiotarsal and ulnacarpal joints.
At the peak of clinical arthritis, mice were sacrificed and the joints were
fixed in Schaffer's
solution, embedded in plastic 9100 and stained with hematoxilin eosin.
Group clinical signs (number of swollen joints/total) on days
13 14 15 16 17 20
------------------ ---------------------------- - -- - -- - --------- - - -----
------ - -- -
Control 0/30 0/30 0/30 0/30 0/30 0/30
SCID, no IL-6 6.5/36 12.5/36 15/36 21/36 30/36 35/36
9ow.arthrit. 189'0 359b 429b 5896 8396 9796
SCID, IL-6 treated 4/30 3.5/30 11/30 7.5/30 12J30 10.5/30
4b w. arthrit. 13 % 12% 37 k 25 % 40% 35 9b
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SCII) mice which are not treated with IL-6 develop severe arthritis due to
infection with
Borrelia bursdorferi starting around day 13 after antigen injection. A low
dose of rh IL-6
reduces the severity of arthritis by an average of 60 - 75 % in all afflicted
animals.
Example 31: Murine model for septic shock
It was decided to investigate the effects of IL-6 in the mouse endotoxic shock
model
using d-galactosamine sensitized mice, since this is widely used as a model
for human septic
shock. Our methods and result are as follows:
Female OF1 mice weighing 18-22 g, were challenged with a 0.2 ml i.p. injection
of a
PBS solution containing 0.15 mg/kg lipopolysaccharide endotoxin (LPS) and 500
mg/kg d-
galactosamine. Mice were divided in groups of 10 mice each and treated as
follows:
Experiment I
Time 11:00 14:00 16:00
Group 1: PBS LPS + d-GAL PBS
Group 2: IL-6 (50 pg) LPS + d-GAL PBS
Group 3: PBS LPS + d-GAL + IL-6 (50 Ng) PBS
Group 4: PBS LPS + d-GAL IL-5 (50 g)
Experiment 2
Time 11:00 14:00 16=00
Group 1: PBS LPS + d-GAL PBS
--Graup 2: IL-6 (50 Ng) LPS +-d-GAL PBS
Group 3: PBS LPS + d-GAL + IL-6 (100 pg) PBS
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Group 4: PBS LPS + d-GAL + IL-6 (20 pg) PBS
Group 5: PBS LPS + d-GAL + IL-6 (5 pg) PBS
Group 6: PBS LPS + d-GAL + IL-6 (0.8 g) PBS
Group 7: PBS LPS + d-GAL + IL-2 (100 pg) PBS
Group 8: PBS LPS + d-GAL + IL-4 (50 pg) PBS
Group 9: PBS LPS + d-GAL IL-6 (50 pg)
rhIL-6 (ILS 969, Sandoz), rhIL-2 (Sandoz) and rhII.-4 (Sandoz) were diluted in
PBS. All
injections (0.2 ml volume) were given intraperitoneaAy. In group 3 (exp. 1)
and group 3 to 8
(exp. 2) IL-6 and IL-2 were diluted into the LPS/d-GAL solution so that mice
received a
single 0.2 ml injection. Numbers in parenthesis indicate the dose of
interleukin given to each
mouse. The multiple dosing of PBS was required to control inter-group
variability due to
stress induced responses due to handling at different times prior or post LPS
challenge.
Mouse survival was observed for 48 hours. For statistical calculation, we used
the Chi
square test. As shown in Figure 1, after 24 hours from LPS challenge, 9 out of
10 control
mice died. IL-6 treatment 3 hours prior to LPS injection or 2 hours after LPS
after LPS
injection, reduced the mortality respectively to 60% (p = 0.12) and 70% (p =
0.26). On the
other hand. IL-6 given at the time of LPS challenge reduced the group
mortality to 10% (p<
0.01). The protective effets were long lasting, since after 48 hours the
mortality in group 3
increased slightly , i.e. to 30%, still indicating a highly significant
protection respect to the
control group (p<0.01). The mortality of group 4 passed from70% to 80%,
whereas no
changes were observed in group 1 and 2.
Based on these results, we tested the effect of II.-6 at different doses. We
gave IL-6
at the time of LPS injection, since according to the first experiment this was
the optimal
--t-ime. We explored as well the effect of..IL-3 and IL-4 given--at the time
of.LPS-as a way to
exclude possible artifacts due to the use of recombinant proteins in the LPS/d-
GAL
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~
preparation. We also tested whether IL-6 was effective in protecting miee from
endotoxic
death a dose of 100 pg/mouse given before or after LPS.
The results of experiment 2(Figun 2) are in line with those of experiment 1.
Also in
this experiment, treatment with IL-6 protected mice from endotoxic death. When
IL-6 was
given together with LPS, the resulting protection 24 hours after LPS was dose
dependent at
the dose of 20 (30% deaths, p = 0.03), 4(5096 deaths, p = 0.16) and 0.8 (70%
deaths, p
0.61) pg/mouse, whereas at the dose of 100 g/mouse (60% deaths, p = 33) mice
were
protected less efficiently than at a 20 pg/mouse. Pre- or post- treatment with
100 pg IL-6 /
mouse resulted in a protection comparable to that observed when the same dose
of IL-6 was
given together with LPS. Similar mouse survival results were obtained 48 hours
after LPS.
IL-4 given at the time of LPS challenge was ineffective in protecting mice
from
endotoxic death, whereas IL-2 decreased mouse survival.
