NZ620392B2 - Biodegradable, semi-crystalline, phase separated, thermoplastic multi block copolymers for controlled release of biologically active compounds - Google Patents
Biodegradable, semi-crystalline, phase separated, thermoplastic multi block copolymers for controlled release of biologically active compounds Download PDFInfo
- Publication number
- NZ620392B2 NZ620392B2 NZ620392A NZ62039212A NZ620392B2 NZ 620392 B2 NZ620392 B2 NZ 620392B2 NZ 620392 A NZ620392 A NZ 620392A NZ 62039212 A NZ62039212 A NZ 62039212A NZ 620392 B2 NZ620392 B2 NZ 620392B2
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- New Zealand
- Prior art keywords
- polymer
- crystalline
- block copolymer
- semi
- biodegradable
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- 239000000622 polydioxanone Substances 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
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- 239000012146 running buffer Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- 101700082413 tant Proteins 0.000 description 1
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Abstract
The disclosure is directed to a biodegradable, semi-crystalline, phase separated thermoplastic multi-block copolymer, a process for preparing said multi-block copolymer, a composition for the delivery of at least one biological active compound, and to o a method for delivering a biologically active compound to a subject in need thereof. A multi-block copolymer of the invention is characterised in that: a) it comprises at least one hydrolysable pre-polymer (A) segment and at least one hydrolysable pre-polymer (B) segment, b) said multi-block copolymer having a T of 37 °C or less and a Tm of 110-250 °C under physiological conditions; c) the segments are linked by a multifunctional chain-extender; d) the segments are randomly distributed over the polymer chain; e) at least part of the pre-polymer (A) segment is derived from a water-soluble polymer. compound to a subject in need thereof. A multi-block copolymer of the invention is characterised in that: a) it comprises at least one hydrolysable pre-polymer (A) segment and at least one hydrolysable pre-polymer (B) segment, b) said multi-block copolymer having a T of 37 °C or less and a Tm of 110-250 °C under physiological conditions; c) the segments are linked by a multifunctional chain-extender; d) the segments are randomly distributed over the polymer chain; e) at least part of the pre-polymer (A) segment is derived from a water-soluble polymer.
Description
Title: RADABLE, SEMI-CRYSTALLINE, PHASE SEPARATED,
THERMOPLASTIC MULTI-BLOCK MERS FOR CONTROLLED
E OF BIOLOGICALLY ACTIVE COMPOUNDS
The invention is directed to biodegradable, semi-crystalline, phase ted,
thermoplastic multi-block copolymers, a process for preparing said multi-block copolymers,
composition for the delivery of at least one biologically active nd, and to methods for
delivering a biologically active compound to a subject in need f.
Peptides and proteins, er called polypeptides, play a vital role in all
biological ses and have received a growing ion in recent years as drug candidates.
The rapid advances in peptide and n pharmacology along with the large-scale production
of these compounds by recombinant DNA technology – among other techniques – have fuelled
enormous interest in these compounds. Unfortunately, peptide and protein development has
far outpaced the ability to deliver these compounds systemically or locally using convenient
and effective delivery systems.
Biodegradable polymers have received increased attention over the past decade for
use in long-acting parenteral controlled release systems, either for systemic or site-specific
drug delivery. Biodegradable controlled release formulations can significantly improve the
pharmacokinetics of therapeutic compounds. This is especially relevant in the treatment of
chronic diseases and for compounds with a narrow therapeutic window since systemic plasma
concentrations can be reduced with concurrent reduction in undesirable side effects. Also
many new biologically active compounds have short half-lives, necessitating nt injection
to achieve therapeutically effective plasma . Patient compliance and the high costs
associated with frequent dosing regimens for parenterally stered biologically active
nds have sed the interest in biodegradable parenteral controlled release dosage
forms.
Poly(D,L-lactic acid) (PDLLA) and copolymers of lactic acid and glycolic acid, also
known as PLGA copolymers, are the most widely d biodegradable rs for use in
parenteral controlled release depot formulations. PLGA copolymers have been successfully
used for the development of sustained release depot formulations for small molecules, such as
risperidone, and therapeutic peptides such as leuprolide, goserelin or octreotide.
PLGA rs have, however, several drawbacks that limit their use and make
them less suitable for the delivery of polypeptides. Firstly, PLGA copolymers are relatively
hydrophobic polymers and do not provide an l environment for encapsulated proteins.
Proteins may adsorb to the polymer, resulting in slow and incomplete release, protein
unfolding and/or aggregation. Secondly, the ability to manipulate the release of larger
biologically active compounds such as an ulated polypeptide is limited since diffusion of
such compounds in the relatively rigid and non-swellable PLGA matrices is negligible. The
release of proteins from PLGA copolymers therefore depends on diffusion via pores present in
the matrix and on the degradation or dissolution time of the matrix. Typically, the
encapsulated protein remains entrapped in the polymer matrix until the moment the latter
has degraded to such an extent that it loses its integrity or dissolves, resulting in biphasic or
triphasic degradation-dependent release profiles typically ed for PLGA-based depot
formulations. y, during degradation of PLGA copolymers, acidic moieties are formed that
accumulate in the rigid and non-swellable PLGA matrix resulting in the formation of an acidic
micro-environment in the polymer matrix with in situ pHs that can be as low as 1-2. Under
such acidic conditions encapsulated proteins may form aggregates g to incomplete
protein release. er, the low pH may have a deleterious effect on the structural integrity
and biological activity of the encapsulated peptide or protein, ially leading to reduced
therapeutic efficacy and enhanced immunogenicity. Chemical modification of proteins and
peptides, such as acylation and adduct formation have been reported.
Thus, there is a need for biodegradable polymers that are more suitable for
protein delivery. r, one of the advantages of PLGA and related polymers is that they
have a proven track record of al use and are generally considered as highly
biocompatible, and as a consequence and e of risk mitigation reasons, have been
adopted by pharmaceutical companies to develop depot formulations for their active
compounds. It is therefore desired that a new biodegradable polymeric protein delivery system
would be designed of polymers that are composed of monomers that are well-known,
biologically safe and clinically acceptable.
In an attempt to provide a hydrophilic matrix with an improved ibility for
protein drugs that allows controlled release thereof, Kissel et al. (J. Contr. Rel. 1996, 39(2),
315-326) synthesised ABA triblock copolymers containing hilic poly(ethylene oxide) B
blocks and hydrophobic, biodegradable A blocks, consisting of poly(L-lactic-co-glycolic acid).
Kissel et al. reported sustained release of various proteinaceous compounds from pheres
composed of poly(L-lactic-co-glycolic acid)―poly(ethylene glycol)―poly(L-lactic-co-glycolic acid)
and poly(L-lactic acid)―poly(ethylene glycol)―poly(L-lactic acid) copolymers, i.e. ABA type
polymers, where A is a hydrophobic block and B is hylene glycol. These copolymers are,
however, limited in their A/B ratio, i.e. poly(ethylene glycol) (PEG) content. To prevent renal
clearance issues associated with the use of high lar weight PEG, the molecular weight
of the PEG moiety used in these ABA triblock copolymers should preferably not exceed 5000
g/mol. Thus, to obtain high PEG content with the triblock polymers described by Kissel et al.
while maintaining low PEG molecular weight, the hydrophobic blocks should also be short.
This would yield rs with undesirable properties for use as biomaterials, as at short
block lengths glass tion temperature (Tg) is below room temperature (determined by the
inventors) and llinity (in case of -lactic acid) (PLLA)) is very low or absent (De
Jong, Macromolecules, 1998, 31(19), 6397-6402), thus yielding sticky als and (too) fast
and poorly controlled release of the incorporated active.
Examples of phase separated, segmented/block copolymers are found in e.g.
US-A-5 554 170, US-A-5 066 772, US-A-5 236 444, US-A-5 133 739, and US-A-4 429 080.
40 These known materials are bioresorbable co-polyesters wherein the hard blocks are
predominantly built of crystalline poly-glycolide and/or poly-lactide. These polymers are rigid
and non-swellable and therefore suffer from the same disadvantages and limitations as
ned for PLGA and PDLA making them unsuitable for the sustained release of ns.
Biodegradable multi-block copolymers ning one hydrolysable polyester
segment and one hydrophilic hydrolytically stable segment have been studied for their drug
loading and release capacity (e.g. multi-block copolymers based on e-caprolactone ts
and poly(ethylene glycol) segments are described by Lee et al. (J. Control. Rel., 2001, 73(2),
315-327). These rs contain only one degradable segment, thus limiting the ability to
control their degradation and release properties.
Known multi-block copolymers of two types of biodegradable lymers
(segments) on the other hand, can only be made in an alternating pre-polymer sequence,
resulting in a d range of possible variables (Penco et al., J. Appl. Polym. Sci. 2000,
78(10), 1721-1728).
Examples of biodegradable multi-block copolymers containing hydrolysable
polyester t of different composition are described in WO-A-2004/007588. These
multi-block copolymers comprise biodegradable phase separated copolymers with segments of
an amorphous, “soft”, biodegradable pre-polymer (A) having a Tg (glass transition
temperature) below 37 °C and segments of a semi-crystalline, “hard”, biodegradable
lymer (B) having a phase transition temperature of 40-100 °C, in which the segments
are linked by a multifunctional chain extender. To obtain multi-block copolymers with Tm of
40-100 °C as disclosed in WO-A-2004/007588, the choice for pre-polymers to be used as B
segments is limited to pre-polymers composed of either poly(e-caprolactone) (PCL)
(WO-A-2004/007588), poly(valerolactone) (PVL) and/or polydioxanone (PDS). When PDS is
used as segment B, multi-block copolymers with a Tm of 80-90 °C 5 711 958) are
obtained. When PCL is used as segment B, multi-block copolymers with a Tm of 40-60 °C
(WO-A-2004/007588) are obtained. PVL homopolymers have a Tm r to PCL
homopolymers (i.e. ~60 °C). Thus, when PVL would be used as segment B, multi-block
copolymers with a Tm of 40-60 °C would be obtained. PDS, PCL and PVL have vely low
Tgs of -10, -60 and -60 °C, respectively. The low Tg of the PDS, PCL and PVL segments limit
the Tg range of the multi-block copolymer (where the Tg originates from phase mixing of the
ous segment A and the amorphous part of the rystalline segment B) that can be
obtained and thus limit the control over e and degradation properties.
WO-A-99/02168 describes biodegradable multi-block mers for biomedical
applications, where either ABA or AB type of pre-polymers are chain-extended.
Chain-extension of either ABA of AB type of pre-polymers can only lead to alternating
multi-block copolymers. An alternating block copolymer is represented by ABABABABAB in
case of chain-extension of AB pre-polymers, or ABAABAABAABA in case of extension of
ABA pre-polymers.
Biodegradable phase separated multi-block copolymers containing a hard and a
soft segment have been described in US-A-6 160 084. This document describes the use of
40 PCL―PLLA multi-block copolymers composed of pre-polymers that are linked with
trimethylhexane-1,6-diisocyanate (THDI). These materials are mentioned to be useful in drug
delivery systems where shape-memory is required. 006/0 140 999 bes the use of
similar shape-memory polymers for use in drug release systems, wherein the shape-memory
material comprises units, derived from monomers selected from the group ting of
caprolactone, lactide, glycolide and dioxanone. Examples include PDS―PCL and PDS―PLGA
multi-block copolymers. These materials cannot exhibit any significant swelling capacity
under (simulated) physiological conditions, since swelling would induce loss of mechanical
properties and thus loss of the memorised shape.
Other phase-separated, segmented multi-block copolymers e polyetherester
copolymers as bed in US-A-5 980 948. These copolymers consist of crystalline aromatic
segments and soft ntaining segments linked by hydrolysable ester bonds. The
copolymers have the inherent drawback that low swellable compositions, i.e. itions rich
in hydrophobic aromatic segments, are not well-degradable, due to the high crystallinity and
hydrophobicity of the aromatic segments. Highly swellable compositions, i.e. compositions rich
in PEG, are not well-degradable either, due to the low concentration of ester bonds. In
contrast, the multi-block copolymers of the current invention are degradable at each segment
A/segment B ratio, due to the presence of ester bonds in segment A as well as in segment B.
Further, in contrast to the multi-block copolymers of the current invention, the Tg of
polyetherester copolymers cannot be varied and is always low, around the Tg of PEG, i.e.
-30 °C.
Objective of the invention is to overcome one or more of the cks observed in
the prior art and/or to provide the public with a useful choice.
In a first aspect the invention is directed to a biodegradable, rystalline
phase separated, thermoplastic multi-block copolymer, the copolymer being characterised in
that:
a) it comprises at least one hydrolysable lymer (A) segment and at least one
hydrolysable pre-polymer (B) t;
b) said multi-block copolymer having a Tg of 37 °C or less and a Tm of 110-250 °C under
physiological conditions;
c) the segments are linked by a multifunctional chain-extender;
d) the segments are randomly distributed over the polymer chain; and
d) at least part of pre-polymer (A) is d from a water-soluble r.
In a second aspect, the invention provides a process for preparing a biodegradable,
semi-crystalline, phase separated, thermoplastic multi-block copolymer ing to the
invention, comprising
i) performing a chain extension reaction of pre-polymer (A) and pre-polymer (B) in the
presence of a multifunctional chain-extender, wherein lymer (A) and (B) are both
diol or diacid terminated and the chain-extender is di-carboxylic acid or diol ated;
ii) performing a chain extension reaction using a coupling agent, wherein pre-polymer (A)
40 and (B) are both diol or diacid terminated and the coupling agent is dicyclohexyl
carbodiimide.
In a third aspect, the invention provides a biodegradable, semi-crystalline, phase
separated, thermoplastic multi-block mer prepared by a method of the invention.
In a fourth aspect, the invention provides a use of a biodegradable, semicrystalline
, phase separated, thermoplastic multi-block copolymer according to the invention
in the manufacture of a medicament.
In a fifth , the invention provides a composition for the delivery of at least
one biologically active compound to a host, comprising at least one biologically active
nd encapsulated in a matrix, wherein said matrix comprises at least one
biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer
according to the invention.
In a sixth aspect, the invention provides a use of a ition according to the
invention, in the cture of a medicament for treating rheumatoid arthritis, hepatitis,
diabetes, metabolic syndromes, osteoarthritis, renal disease, inflammation, local pain
processes, local infections, local skin diseases, tumours (or their sites after surgical removal as
a postoperative treatment to destroy any tumour cells possibly ing), prostate or breast
cancer, galy, ocular diseases such as age-related macular degeneration, local brain
diseases such as son’s disease, and cardiovascular diseases such as acute myocardial
tion, chronic heart failure or arthrosclerosis.
In a seventh aspect, the invention provides a method of manufacturing a
composition according to the invention, comprising the successive steps of
a) fying an aqueous on of a water-soluble biologically active compound in a
on of a biodegradable, semi-crystalline, phase separated, thermoplastic multi-block
mer according to the invention in an organic solvent;
b) subsequently emulsifying the resultant emulsion of a) in an aqueous solution sing
a surfactant, thereby yielding a in-oil-in-water (W/O/W) emulsion; and
c) extracting the organic solvent to solidify microspheres.
In an eighth aspect, the invention provides a method of manufacturing a
composition according to the invention, comprising the successive steps of
a) dispersing the biologically active compound as a solid powder in a solution of a
biodegradable, rystalline, phase separated, thermoplastic multi-block copolymer
according to the invention in an organic solvent;
b) emulsifying the resultant dispersion of a) in an aqueous solution comprising a
tant, thereby yielding a solid-in-oil-in-water (S/O/W) emulsion; and
c) extracting the organic solvent to solidify the microspheres.
In a ninth aspect, the invention provides a method of manufacturing a composition
according to the invention, comprising the successive steps of
a) emulsifying an s solution of a water-soluble biologically active compound in a
solution of a biodegradable, semi-crystalline, phase separated, plastic multi-block
copolymer according to the invention in an c solvent;
40 b) adding a polymer precipitant, to the resultant emulsion of a) to form embryonic
microparticles; and
c) extracting the polymer precipitant and the organic solvent to solidify the microspheres.
In a tenth aspect, the invention es a method of manufacturing a composition
according to the invention, comprising the successive steps of
a) sing the biologically active compound as a solid powder in a solution of a
radable, semi-crystalline, phase separated, thermoplastic multi-block copolymer
according to the invention in an organic solvent;
b) adding a polymer precipitant, to the resultant dispersion of a) to form embryonic
microparticles; and
c) extracting the polymer itant and the organic solvent to solidify the microspheres.
In an eleventh aspect, the invention provides a composition ed by the
methods of the invention.
The multi-block copolymer of the invention can be composed of at least two
different ts each having different physical characteristics, including degradation and
ng characteristics. Due to their unique make-up and their semi-crystalline phase
separated morphology, the materials of the invention are surprisingly versatile and extremely
suited for constructing drug delivery matrices and drug eluting coatings, which are utilisable
for encapsulating certain therapeutic agents and for sustained release of the encapsulated
therapeutic agent either locally or into the systemic circulation. As is described herein below,
the composition of the invention is of particular interest for the controlled e of a
biologically active compound, such as a biologically active polypeptide to a host.
The term “phase-separated” as used herein is meant to refer to a system, in
particular a copolymer, built of two or more different pre-polymers, of which at least two are
ally) incompatible with each other at body temperature or below (under physiological
conditions such as in the human body). Thus the pre-polymers do not form a homogeneous
e when combined, neither when combined as a physical mixture of the pre-polymers,
nor when the lymers are combined in a single chemical species as “chemical mixture”,
viz. as copolymer.
The term “pre-polymer” as used herein is meant to refer to the polymer segments
that are randomly linked by a multi-functional chain extender, together making up the
multi-block copolymer of the invention. Each lymer may be obtained by polymerisation
of suitable monomers, which monomers thus are the chemical units of each pre-polymer. The
d properties of the pre-polymers and, by consequence, of the block copolymer of the
invention, can be controlled by choosing a pre-polymer of a suitable composition and molecular
weight (in particular Mn), such that the required Tm or Tg is obtained.
The term “multi-block” as used herein is meant to refer to the ce of at least
two ct pre-polymer segments in a polymer chain.
The term “thermoplastic” as used herein is meant to refer to the non cross-linked
nature of the multi-block copolymer. Upon heating, a thermoplastic polymer becomes fluid,
whereas it solidifies upon (re-)cooling. Thermoplastic rs are soluble in proper solvents.
40 The term “hydrolysable” as used herein is meant to refer to the ability of reacting
with water upon which the molecule is cleaved. Hydrolysable groups e ester, carbonate,
phosphazene, amide and urethane groups. Under physiological conditions, only ester,
carbonate and phosphazene groups react with water in a reasonable time scale.
The term “multifunctional chain-extender” as used herein is meant to refer to the
presence of at least two reactive groups on the chain-extender that allow chemically linking
ve pre-polymers thereby forming a multi-block copolymer.
The term “random multi-block copolymer” as used herein is meant to refer to a
multi-block copolymer where the distinct segments are distributed randomly over the polymer
chain.
The term “water-soluble polymer’’ as used herein is meant to refer to a polymer
that has a good solubility in an aqueous medium, preferably water, under physiological
ions. This polymer, when copolymerised with more hydrophobic moieties, renders the
resulting copolymer swellable in water. The water-soluble r can be derived from a diol,
a e or a diacid. The diol or diacid is suitably used to initiate the ring-opening
polymerisation of cyclic monomers.
The term “swellable” as used herein is meant to refer to the uptake of water by the
polymer. The swelling ratio can be calculated by dividing the mass of the swollen
copolymer by that of the dry copolymer.
The term “semi-crystalline” as used herein is meant to refer to a morphology of the
multi-block copolymer that comprises two distinctive phases, an amorphous phase and a
crystalline phase. Preferably, the multi-block copolymer is made up of an amorphous phase
and a crystalline phase.
The term “biologically active compound” as used herein is ed to be broadly
interpreted as any agent that provides a eutic or prophylactic effect. Such agents
include, but are not limited to, antimicrobial agents (including antibacterial and antifungal
agents), anti-viral agents, anti-tumour agents, hormones and immunogenic agents.
The term “biologically active polypeptide” as used herein is meant to refer to
es and proteins that are biologically active in a mammal body, more in particular in the
human body.
The term “comprising” as used in this specification and claims means “consisting
at least in part of”. When interpreting statements in this specification, and claims which
include the term “comprising”, it is to be understood that other features that are additional to
the features prefaced by this term in each statement or claim may also be t. Related
terms such as “comprise” and “comprised” are to be reted in similar manner.
The semi-crystalline, phase separated multi-block copolymers of the invention
me one or more of the aforementioned drawbacks and limitations. Due to the presence of
segments derived from a water-soluble r (such as hydrophilic PEG segments), the phase
separated multi-block copolymer swells in an aqueous environment to form a swollen hydrogel
providing a natural environment for biologically active compounds such as proteins. When the
multi-block copolymers of the ion are applied as a polymer matrix in a controlled release
40 ation for delivering a biologically active nd, the swellability of the multi-block
copolymers can avoid accumulation in the r matrix of acidic degradation products
formed during hydrolysis of the polymer chains. Instead, such ation products are
released from the matrix and thereby prevent the ion of an acidic micro-environment in
the polymer matrix that would be deleterious to the ulated biologically active
nd. Moreover, due to the swellability of the phase separated multi-block copolymers of
the invention, any encapsulated compounds can be released gradually by diffusion thereby
preventing the biphasic or triphasic e patterns lly obtained for non-swellable
biodegradable polyesters such as poly(D,L-lactide) or actic-co-glycolic acid).
The multi-block copolymers of the invention have a Tm of 110-250 °C under
physiological conditions. This is due to the pre-polymer segment B. The segment B is based on
crystallisable rs, such as PLLA, poly(D-lactic acid) (PDLA), polyglycolic acid (PGA) or
polyhydroxybutyrate (PHB), or combinations of crystallisable polymers. Most ably, the
segment B is based on a pre-polymer composed of PLLA. The amorphous phase of the phase
separated multi-block copolymers of the invention predominantly ts of the soft A
segments. Surprisingly, we have found that the amorphous part of the hard segments B also
contribute to the total amorphous phase of the multi-block copolymers of this invention.
For multi-block copolymers described in WO-A-2004/007588 the choice for
pre-polymers to be used as B segments is limited to pre-polymers composed of
poly(e-caprolactone) (PCL), poly(valerolactone) (PVL) and poly(dioxanone) (PDS) due to the Tm
of pre-polymer (B) being in the range of 40-100 °C (regarding common polyesters used for
biomedical applications). In accordance with the invention, the Tm of pre-polymer (B) is
preferably in the range of 110-250 °C. As a result, pre-polymer (B) can be selected from a list of
chemically different pre-polymers that were not previously considered. The inventors found
that the different chemistry for pre-polymer (B) yields multi-block copolymers that exhibit
advantageous properties which cannot be ed with the mers described in
WO-A-2004/007588.
When PDS is used as segment B, multi-block copolymers with a Tm of 80-90 °C
(US-A-5 711 958) are obtained. When PCL is used as segment B, multi-block copolymers with
a Tm of 40-60 °C (US-A-5 711 958) are obtained. PVL homopolymer has a Tm of approximately
60 °C, similar to PCL homopolymer. When PVL segments are used as segment B, multi-block
copolymers with a Tm of approximately 40-60 °C are obtained. PDS, PCL and PVL are
semi-crystalline, and thus s a Tg in addition to their Tm. PDS, PCL and PVL all possess
a low Tg of their respective amorphous phases of approximately -10 °C, -60 °C and -70 °C,
respectively. Increasing the temperature range for block B to 110-250 °C opens up the
possibility to use PLLA, PDLA, PGA and PHB. These polymers have a higher Tg of
approximately 50 °C, 35 °C and 0 °C, respectively. Irrespective of which r is used for
the hard B segments, these hard B segments will always be semi-crystalline by themselves, i.e.
partly ous. Surprisingly, it was found that the amorphous part of the hard B segments
will (partly) phase mix with the soft A segments and thus both will contribute to the overall Tg
of the multi-block mer. ore, the Tg of the amorphous phase is determined by both
40 the Tg of segment A and the Tg of t B, in combination with the molar ratio of segment
A/B. The Tg can be varied from Tg close to pre-polymer (A) (when pre-polymer A/B ratio of close
to 1 is used) to Tg close to pre-polymer B (when pre-polymer A/B ratio close to zero is used).
Importantly, the release of actives encapsulated in the polymer matrix s heavily on the
Tg of the amorphous phase, as the diffusion of actives occurs through the amorphous phase
and not the dense, crystalline phase. Also, the ation rate of a polymer depends heavily
on the Tg of the amorphous phase, as this influences the rate of water influx and thus the rate
of hydrolysis. The use of pre-polymer (B) with Tm 0 °C having relatively high Tg enables
covering a much broader Tg range than would have been possible with pre-polymer (B) having
Tm 40-100 °C and a relatively low Tg. As a consequence, the using such pre-polymers (B) for
ing multi-block copolymers with a Tm in the range of 110-250 °C enables a much broader
range of release and degradation properties of the polymer and thus also allows better control
over the release of different biologically active compounds.
Furthermore, the higher Tm of the multi-block copolymers of the current invention
allows the preparation of non-sticky microspheres by a double-emulsion process at ambient
conditions, while still having short B ts. The limitation of the length of the
crystallisable B segment is important to have multi-block copolymers that degrade well under
physiological conditions, contrary to higher lar weight lline PLLA polymers. In
contrast, pheres cannot be made using multi-block copolymers where segment B is
composed of a short PCL since the short PCL blocks do not form crystalline domains during
microsphere formation. As a consequence the polymer remains amorphous. Due to the low Tg
of the amorphous polymer, the polymer is sticky due to which microspheres erate and
fuse together during the extraction / evaporation process step. Since PVL has a similar Tm as
PCL, it is to be expected that microspheres cannot be made using multi-block copolymers
where t B is composed of a short PVL pre-polymer. No reference has been made in
literature of microspheres composed of PDS or PDS copolymers. It is known from ture
that crystallisation of PDS is slow and incomplete at fast cooling rates and/or low PDS
molecular weight. These results predict that preparation of microspheres by a double-emulsion
s using multi-block copolymers with segment B being a short PDS block is not feasible.
Theoretically, the storage stability of microspheres at ambient conditions made
with pre-polymer (B) having Tm 110-250 °C is improved compared to pre polymer (B) having
Tm 40-100 °C. Increased Tm ses the Tc and thus increases the llinity of the
microspheres. A higher llinity will reduce the molecular mobility of the encapsulated
biologically active compound in the polymer matrix and improve the storage stability of the
product. It is known from literature that increased crystallinity increases the storage stability
of particles. Also, pre-polymers B having Tm 110-250 °C have higher Tg compared to
pre-polymers (B) having Tm 40-100 °C. It is known from literature that for semi-crystalline as
well as amorphous particles, increased Tg increases the storage stability.
The block copolymers of the invention further have an ed
degradation rate compared to multi-block mers where the crystallisable t is
based on PCL, because the B segments in the multi-block copolymers of the invention are less
40 hydrophobic compared to PCL.
Synthesis of multi-block copolymers where the crystallisable segment is based on
PDS is ed by the limited risation of the PDS monomer, p-dioxanone and the
limited lity of PDS in common solvents. It is well known that p-dioxanone has a
relatively low ceiling temperature, leading to maximum conversion of imately 80 %. In
contrast, monomers used for the block copolymers of the invention, such as e and
ide, can be easily polymerised to conversions above 95 %. The limited solubility of PDS
containing polymers also limits their use for ation of controlled release formulations.
Multi-block copolymers of this invention that are composed of a PLLA-based
segment B have the additional advantage that PDLA may be added as an additional B
segment, yielding multi-block copolymers with increased crystallinity and decreased
degradation rate due to the formation of PLLA/PDLA stereocomplex crystals with a Tm as high
as 220 °C, which is approximately 50 °C higher than the Tm of crystalline PLLA segments that
are solely composed of omer L-lactide.
In the multi-block copolymers of the invention, the content of the ts
derived from a water-soluble polymer may be varied independently from the block length of
the hydrophobic (crystalline) segment. Therefore, high contents of segments that are derived
from a water-soluble r can be obtained, while maintaining llinity. Furthermore,
the intrinsic viscosity (IV) of the multi-block copolymers of the invention may be varied
independently from the composition, in contrast to the ABA triblock copolymers described by
Kissel et al. The high degree of variability of the multi-block copolymers of the invention
allows easy tuning of the , ratio and composition of the segments to obtain the desired
degradation teristics and drug release kinetics.
The multi-block copolymers of this invention further have advantages over the
block copolymers of structure ABA as mentioned in the examples of the introduction. Although
polymer properties can be greatly ed by using block copolymers with blocks of different
copolymers instead of homo or random copolymers, these ABA copolymers still have certain
disadvantages.
To obtain a minimum molecular weight of the ABA copolymer, the sequences A
and B must have a certain length. The blocks may independently behave as the individual
homopolymers with similar ition. Properties of the ABA type copolymers can only be
tuned by varying the composition of A and B blocks. Another antage is that block
copolymers must be prepared at relatively high temperatures (> 100 °C) under inert ions
for complete conversion of all the monomers and to obtain sufficient molecular weight. The
first disadvantage can be solved by using multi-block copolymers wherein the blocks or
segments are much shorter and linked together by a chemical reaction performed at
atures below 100 °C. Properties such as degradation behaviour can be tuned in a much
better way by choosing the proper combination of segment lengths, ratio and composition.
Furthermore, due to the relatively high temperatures used in the process of
preparing ABA block copolymers (and derivatives thereof), there is always a possibility of
40 transesterification, resulting in a certain extent of phase mixing. The multi-block mers
of the invention do not suffer from this disadvantage since they can be prepared by linking
pre-polymers with previously determined r composition at rather low temperatures
(< 100 °C) thus avoiding transesterification and other side-reactions reactions, which may
cause the generation of undesired degradation and other by-products. This means that the
monomer sequence length of the copolymer is determined by the choice of building components
and not so much by on time and temperature, as being y applied for synthesis of
random mers. Another age of multi-block copolymers of this invention prepared
by g of pre-polymers using a multifunctional chain-extender is that the pre-polymer
ts are randomly distributed in the copolymer, thus offering much more possibilities of
tuning the properties. A random multi-block copolymer is for example
ABBBBABAAABBAAAAA... etc. The random multi-block copolymers of the invention e
many ages that cannot be obtained with alternating multi-block copolymers.
Firstly, the random multi-block copolymers obtained by chain extension of A and
B blocks have an unlimited A to B ratio. A:B can, for instance, be 10:90, but may as well be
90:10. In st, the ratio of the blocks in an ating block copolymer is limited to
the ratio used in the chain extended polymer. For instance, in the case of chain extension of
AB the A:B ratio in the multi-block copolymer is 50:50. The random nature of the multi-block
copolymers of the invention greatly increases the possible compositions of the material and
thereby the control over its physical and al ties. This includes a better control of
the swelling capacity in water, morphology (phase tion, amorphous/crystallinity) and
polymer ation.
Secondly, the synthesis method of the random multi-block copolymers of the
invention is much less laborious as compared to the synthesis of alternating multi-block
copolymers. In alternating multi-block copolymers either segments A and B in case of AB
diblocks, or segments A and C in case of ACA triblocks, have to be linked prior to
chain-extension (or a macro chain-extender needs to be synthesised). In random multi-block
copolymers, separate A and B blocks are chain extended with e.g. a commercially available
extender.
Another advantage of the multi-block copolymers of the invention is that they are
based on a multifunctional (preferably aliphatic) chain-extender. By choosing the type and
amount of chain-extender the polymers properties can be affected (for instance, the
chain-extender may act as a softener or it may affect the degree of phase separation). The total
degree of m to obtain polymers with the desired properties is therefore increased
compared to polymers of the prior art.
In accordance with the invention phase separated multi-block copolymers are
provided that swell sufficiently in an aqueous environment and under physiological conditions
upon administration so as to provide an aqueous microenvironment for the encapsulated
peptide or protein and allow diffusion controlled release of the peptides and proteins. The
materials thus show a significant decrease of the mechanical strength. Although such
materials can be used as shape-memory materials under dry conditions without showing a
40 significant decrease in mechanical strength prior to the transition to the memorised shape, e.g.
by means of using temperature or light as an external trigger, these materials do show
significant dimensional changes and a significant decrease of their mechanical strength under
hydrated conditions, simply because these materials absorb significant amounts of water due
to their hydrophilic character leading to extensive swelling and plasticisation of the material.
As a uence, under hydrated conditions, such as the physiological conditions encountered
in a human or animal body, the size of constructs prepared of these materials changes
significantly and the mechanical properties of these als change orders of magnitude.
Contrary to the multi-block copolymers of the current invention, the shape-memory materials
described in US-A-5 711 958 hardly swell under hydrated conditions, such as the physiological
ions encountered in a human or animal body.
Phase separated polyesters or ter-carbonates of this invention are a
promising group of biomaterials and can be used in various drug delivery applications since
they provide excellent control over drug release and allow release of biologically active
nds, such as polypeptides.
The logy of the multi-block copolymer (or of a construct made thereof) is
dependent on the nmental conditions: a DSC (Differential Scanning Calorimetry)
measurement may be med under inert (dry) conditions and the s may be used to
determine the dry materials’ thermal properties. However, the logy and ties
under physiological conditions (i.e., in the body) may be different from the morphology and
properties under ambient conditions (dry, room temperature). It is to be understood that the
transition temperatures, Tg and Tm as used herein, refer to the corresponding values of a
material when applied in vivo; viz. when at brium with an atmosphere that is saturated
with water vapour and at body temperature. This may be simulated in vitro by performing the
DSC ement after allowing the material to brate with a water-saturated
atmosphere. When in dry state, the materials used in the invention may have Tg values that
are somewhat higher than at ian body conditions, that is to say, when the dry
materials are subjected to DSC, the first inflection point may arise at higher temperatures, for
instance at 42 °C or 50 °C, or more. Upon ation in vivo, however, the dry material’s Tg
and/or Tm will drop as a result of the absorption of water, which plasticises the polymer and
this final Tg should be around body temperature or lower according to the invention. The final
Tm should be present at temperatures between 110 °C and 250 °C under physiological
conditions.
For instance, a polymer that contains PEG in the soft segment can be crystalline
under dry conditions at ambient temperature, while amorphous under wet conditions, giving a
mixed Tg or two separated Tgs of the soft segment formed by amorphous softened PEG and the
polyester/carbonate. The phase separated character of the copolymers of the ion is
reflected in the profile of the Tg or Tm. The phase separated mers are characterised by at
least two phase transitions, each of which is related to (but in general not identical to) the
corresponding Tg or Tm values of the pre-polymers which are comprised in the copolymer. The
Tg is determined by taking the midpoint of the specific heat jump, as may be measured e.g. by
40 DSC. The Tm is the peak maximum of the melting peak, as is schematically illustrated in
Figure 1, that shows the heat flow endotherm for a copolymer characterised by a Tg and a Tm.
As defined herein, values of Tg and Tm of a certain pre-polymer t the values as measured
on the copolymer. In case of complete immiscibility of the pre-polymers, the Tg of the
copolymer is governed solely by the Tg of the amorphous, “soft” pre-polymer. In practice,
however, the composition of the crystalline and amorphous phase of the multi-block copolymer
is not the same as the ition of the soft A segments and the semi-crystalline B segments.
The amorphous part of the original hard segment forming pre-polymer will mix with the soft
segment forming pre-polymer (A) and thus become part of the amorphous phase. The Tg value
of the amorphous phase is then different from that of the pre-polymer used. The extent of
miscibility (and therefore the deviation of Tg and/or Tm from those of the corresponding
pre-polymers) is dependent on the pre-polymer composition, ratio and segment length in the
copolymer. The Tg of the copolymer segments generally lies between the Tg value of the phase
mixed copolymer and the Tg value of the separate lymers.
The physicochemical properties (such as degradation, ng and thermal
ties) of the multi-block copolymers can be easily tuned by changing the type of
monomers of the soft and hard segment forming lymers and their chain length and
chain ratio and by choosing the type and amount of chain-extender. Furthermore, the phase
transition temperatures are low enough for processing the polymer in the melt. The monomer
ratio and distribution of the copolymer can be easily controlled by varying the polymerisation
conditions.
A crystalline segment B is usually desired to obtain non-sticky materials. Also,
the phase separated morphology, with amorphous and crystalline domains, must be
maintained during exposure to physiological conditions (i.e. an aqueous environment at body
temperature) in order to have controlled swelling of the polymer matrix. Control over the
swelling degree is essential to control the release of encapsulated compounds. The crystalline
B segments act as physical links that control the swelling of the more hydrophilic soft
segments. s being affected by the content of hard segment B, the swelling degree of the
polymers depends on the t and lar weight/length of water-soluble polymer in the
soft A segment.
As mentioned previously, a prerequisite of the phase separated segmented
co-polyester is that the Tm of the ter segment B is in the range of 110-250 °C and the Tg
of the segment A is below 37 °C under physiological conditions. The Tm of segment B in the
multi-block copolymer will in general be lower than that of the non-reacted lymer (B)
due to decreased chain flexibility once the pre-polymer is built in in the multi-block copolymer
and due to possible phase mixing of other components of the multi-block copolymer in the
lline phase. An important class of segmented co-polyesters with good phase separation
are those based on hard ts B composed of crystalline PLLA. The ors have shown
that multi-block copolymers with PLLA-based B segments have Tm of at least 110 °C under
physiological ions. These multi-block copolymers offer several advantages. A wide range
of degradation rate can be ed. Pre-polymer (B) that forms the hard segment B is based
40 on crystalline PLLA and such polymers are known to degrade very . In contrast,
pre-polymer (A) is a r that is based on a water-soluble polymer and amorphous
polyester. Such polymers are known to degrade vely fast. The final degradation rate is
determined by the segment A / segment B ratio and can thus be easily tuned. Since release is
among others ed by the degradation rate of the multi-block copolymer, this can also be
tuned by the segment A / segment B ratio. Also, the crystallinity can easily be increased by
mixing PLLA with PDLA to form a stereocomplex. The complexation leads to higher
crystallinity compared to the single enantiomer and also to higher Tm (~ 50 °C higher than the
single enantiomer). Furthermore, the Tg of multi-block copolymers with PLLA-based B
segments can be varied in a wide range, from about -40 up to 40 °C (measured under dry
conditions). Since degradation rate and release rate are, among others, governed by Tg of the
matrix, this wide Tg range also offers great tuning of the release and degradation properties.
Generally, the desired phase separated logy cted by one melting point
and at least one low Tg value) may be obtained by varying the composition, e.g. by choosing the
number average molecular weight, Mn, of the A and B lymers. It is also possible to
influence the phase separated morphology by varying the segment A/segment B ratio.
The segmented multi-block copolymers of this invention comprise a soft segment A
d from pre-polymer (A) which is hydrolysable and typically completely amorphous at
physiological (body) ions. Furthermore, pre-polymer (A) preferably has at least one
phase transition being a Tg of 37 °C or less, preferably 25 °C or less as measured under
physiological (body) conditions. This t will be part of the amorphous phase in the
multi-block copolymer, wherein the amorphous phase is referred herein as phase (A). The
copolymers of the ion also comprise a hard segment B derived from pre-polymer (B),
comprising a semi-crystalline, hydrolysable polymer typically with a Tm of 110-250 °C as
ed at logical (body) conditions. Segment B mostly contributes to phase (B). The
pre-polymers A and B that form the “soft” and “hard” segments, respectively, are linked by a
multifunctional chain-extender. Typically, the crystalline phase(s) is (are) comprised of hard
segments B and the amorphous phase(s) is (are) comprised of soft segments A and the
amorphous part of segments B. The lline and amorphous phase(s) is (are) incompatible
or only partially compatible at body conditions, viz. they phase separate. The multifunctional
chain-extender is ably an aliphatic molecule.
The resulting multi-block copolymers of the invention preferably have a structure
according to formula (1):
―[R1-H-R1-Q1-R4-Q2]x―[R2-Q3-R4-Q4]y―[R3-Q5-R4-Q6]z― (1)
wherein R1 is part of segment A, which is part of phase (A), and may be
amorphous polyester, amorphous polyetherester or ous polycarbonate; or an
amorphous pre-polymer that is obtained from combined ester, ether and/or carbonate groups.
H is the middle block of segment A and is derived from a water-soluble polymer. The block
derived from the water-soluble polymer may be amorphous or semi-crystalline at room
temperature. However, block H thus introduced in t A will become amorphous at
physiological conditions. This water-soluble polymer is selected from the group consisting of
40 polyethers such as hylene glycol (PEG), polytetramethyleneoxide (PTMO) and
polypropyleneglycol (PPG); polyvinylalcohol (PVA) polyvinylpyrrolidone (PVP),
polyvinylcaprolactam, poly(hydroxyethylmethacrylate) (poly-(HEMA)), polyphosphazenes,
polyorthoesters, polyorthoesteramides or copolymers of the previous polymers. Preferably, H is
PEG, which is the initiator of the ring-opening polymerisation of a cyclic monomer that forms
R2 is segment B and mainly or entirely contributes to phase (B). R2 may be a
crystalline or semi-crystalline polyester, polyetherester, polycarbonate or polyanhydride; or
pre-polymers of ed ester, ether, anhydride and/or carbonate . It is possible that
part of phase R2 is ous, in which case this part of R2 will contribute to phase (A). R1 and
R2 are preferably not the same. The variable z is zero or a positive integer. Variables x and y
are both a positive integer.
Optionally, segment R3 is present. This segment is derived from a water-soluble
r that is chosen from the group of polymers mentioned for H. R3 will be part of the
amorphous phase (A) under logical conditions. If R3 is present then the multi-block
copolymer of the invention comprises a soluble polymer as an additional pre-polymer.
Preferably, this water-soluble polymer is selected from the group consisting of polyethers such
as hylene glycol (PEG), polytetramethyleneoxide (PTMO) and polypropyleneglycol
(PPG); polyvinylalcohol (PVA) polyvinylpyrrolidone (PVP), nylcarprolactam,
poly(hydroxymethylmethacrylate) (poly-(HEMA)), polyphosphazenes, polyorthoesters,
polyorthoesteramides or copolymers of the previous polymers. For example, the said
water-soluble polymeric segment is derived from PEG having a Mn of 150-5000 g/mol.
R4 is derived from the chain-extender and consists of an tic C2-C8 alkylene
group, optionally substituted by a C1-C10 alkylene, the aliphatic group being linear or cyclic. R4
is preferably a butylene, ―(CH2)4―, group. The C1-C10 alkylene side group may contain
protected S, N, P or O moieties. Chain-extenders ning aromatic groups are generally not
le, since chain-extenders containing aromatic groups may give rise to undesired
degradation products. Therefore, aliphatic chain-extenders are preferred.
Q1-Q6 are g units obtained by the reaction of the pre-polymers with the
multifunctional chain-extender. Each of Q1-Q6 may be ndently selected from amine,
urethane, amide, carbonate, ester and anhydride. The event that all linking groups Q are
different is rare and usually not preferred.
Typically, one type of chain-extender may be used with three pre-polymers having
the same end-groups resulting in a mer of formula (1) with six similar g groups.
In case pre-polymers R1 and R2 are differently terminated, two types of groups Q
will be present: e.g. Q1 and Q2 will be the same between two linked segments R1, but Q1 and Q2
are different when R1 and R2 are linked. The examples of formula (1) show the result of the
reaction with a difunctional extender and difunctional pre-polymers.
With reference to formula (1) the polyesters of the invention may also be
represented as multi-block or segmented copolymers having a random distribution of segments
(AB)r, wherein ‘A’ corresponds to the segment A and ‘B’ corresponds to the segment B (for
40 z = 0). In (AB)r, the A / B ratio (corresponding to x / y in formula (1)) may be unity or away
from unity. The pre-polymers can be mixed in any desired amount and can be coupled by a
multifunctional chain-extender, viz. a compound having at least two onal groups by
which it can be used to chemically link the pre-polymers. ably, this is a difunctional
chain-extender. In case z „ 0, then the presentation of a random distribution of all the
segments can be given by (ABC)r were three different pre-polymers (one being a segment
derived from a water-soluble polymer such as PEG) are randomly distributed in all possible
ratios.
The lymers of which the a and b (and optionally c) segments are formed in
(AB)r and (ABC)r are linked by the multifunctional chain-extender. This chain-extender is
preferably a diisocyanate chain-extender, but can also be a diacid or diol compound. In case
the pre-polymers all n hydroxyl end-groups and a diisocyanate extender is used,
the linking units will be urethane groups. In case (one of) the pre-polymers (is) are carboxylic
acid terminated, the linking units are amide groups. Multi-block copolymers with structure
(AB)r and (ABC)r can also be prepared by reaction of di-carboxylic acid terminated
pre-polymers with a diol extender or vice versa (diol terminated pre-polymer with diacid
chain-extender) using a coupling agent such as DCC (dicyclohexyl carbodiimide) forming ester
linkages.
As mentioned above, randomly segmented mers refer to copolymers that
have a random bution (i.e. not alternating) of the segments A and B. In case of segments
A and B this can be represented by (AB)r, in case of segments A, B and C this can be
represented by (ABC)r.
The hydrolysable segment R1-H-R1 of formula (1) is obtained by reaction of
pre-polymer (A).
Pre-polymer (A) may e.g. be prepared by ring-opening polymerisation. Thus a
pre-polymer (A) may be a hydrolysable mer ed by ring-opening polymerisation
initiated by a diol or diacid compound, preferably having a random monomer distribution. The
diol compound is preferably an tic diol or a low molecular weight polyether such as PEG.
The polyether is part of the pre-polymer (A) by using it as an tor and it can additionally
be mixed with the pre-polymer (A), thus forming an onal hydrophilic segment R3 in
formula (1). Pre-polymer (A) may be a hydrolysable polyester, polyetherester, polycarbonate,
polyestercarbonate, polyanhydride or copolymers thereof. For example, pre-polymer (A)
comprises reaction products of ester forming monomers selected from diols, oxylic acids
and hydroxycarboxylic acids. Pre-polymer (A) may comprise reaction products of cyclic
monomers and/or non cyclic monomers. Exemplary cyclic rs include ide, lactide,
e-caprolactone, d-valerolactone, trimethylene carbonate, tetramethylene carbonate,
1,5-dioxepaneone, 1,4-dioxaneone (para-dioxanone) and/or cyclic anhydrides such as
oxepane-2,7-dione. In one embodiment, L-lactide, D-lactide and/or D,L-lactide is used.
To fulfil the ement of a Tg below 37 °C, some of the above-mentioned
monomers or combinations of monomers are more preferred than others. For example,
pre-polymer (A) containing the monomers lactide and/or glycolide is preferably combined with
40 any of the other mentioned cyclic co-monomers rolactone, d-valerolactone,
trimethylenecarbonate, 1,4-dioxaneone and combinations thereof). This may by itself lower
the Tg. Alternatively, the pre-polymer is initiated with a PEG with ient molecular weight
to lower the Tg of the multi-block copolymer.
In case pre-polymer A contains ,L-lactide), the L/D ratio of the lactide may be
away from unity (other than 50/50). For instance, an L/D ratio between 85/15 and 15/85 gives a
completely amorphous homo-polymer. Furthermore, it is known that an excess of one isomer
(L or D) over the other increases the Tg of the poly(D,L-lactide). A minor amount of any other of
the above mentioned rs that build the amorphous phase may also be present in the
crystalline phase forming pre-polymer or block.
Furthermore, pre-polymer (A) can be based on (mixtures of) condensation
(non-cyclic) type of monomers such as hydroxyacids (e.g. lactic acid, glycolic acid,
ybutyric acid), diacids (e.g. glutaric, adipic or succinic acid, sebacic acid) and diols such
as ethylene glycol, lene glycol, 1,4-butanediol or xanediol, forming ester and/or
anhydride hydrolysable moieties.
The segment R2 of formula (1) may be obtained by reaction of pre-polymers (B)
derived from monomers L-lactide, D-lactide, hydroxybutyrate, glycolide or a combination of
these monomers resulting in stereocomplex formation, having a phase transition between
110 °C and 250 °C under physiological conditions Preferably, segment B is obtained by
reaction of L-lactide monomer.
Typically, lymer (B) has an Mn of 1000 g/mol or more, preferably 2000 g/mol
or more, more preferably 3000 g/mol or more. In general, Mn of pre-polymer (B) will 10 000
g/mol or less. The content of pre-polymer (B) in the copolymer is preferably 10-90 wt.% based
on total weight of the multi-block copolymer, more preferably 25-70 wt.%, most preferably
-50 wt.%.
The pre-polymers will preferably be linear and random (co)polyesters,
ter-carbonates, polyetheresters, or polyanhydrides with reactive end-groups. These
oups may be hydroxyl or yl. It is preferred to have a dihydroxy terminated
copolymer, but hydroxy-carboxyl or dicarboxyl terminated polymers can also be used. In case
the r has to be linear, it can be prepared with a difunctional component (diol) as a
starter, but in case a three or higher functional polyol is used, star shaped polyesters may be
obtained. The diol in pre-polymer (A) can be an aliphatic diol or a low molecular weight
her.
The pre-polymer synthesis by a ring-opening polymerisation is preferably d
out in the presence of a catalyst. A suitable catalyst is Sn(Oct)2 with M/I = 5000-30 000 (M/I is
the monomer to initiator ratio). It is also possible to carry out the synthesis without a catalyst.
The conditions for preparing the polyesters, polycarbonates and polyanhydrides
are those known in the art.
The copolymers of the invention are generally linear. r, it is also possible
to prepare the copolymers in a branched form. These non-linear copolymers of the invention
may be obtained by using a trifunctional (or higher functional) chain-extender, such as
40 tri-isocyanate. Branched copolymers may show improved creep characteristics.
For the crystallisable hard segment, the length (Mn) of the pre-polymer must be
large enough to be able to crystallise in the copolymer. For example, PLLA hard segment
forming lymer ably has a Mn of 700 g/mol or more, more preferably 2000 g/mol or
more, most preferably 3000 g/mol or more. A larger PLLA pre-polymer length is expected to
result in a phase separated logy at a lower hard segment content. The pre-polymer
ratio at which phase tion is observed is therefore dependent on the lymer lengths.
In general, the lengths of the pre-polymers that form the soft and hard segment within a
mer must have a value at which a phase separated morphology is observed, the extent of
phase separation (incompatibility) being favourable for the desired ties of the ical
device.
The soft segment forming pre-polymer (A) can have an Mn of 500 g/mol or more,
preferably 1000 g/mol or more, more preferably 2000 g/mol or more. The length of the
pre-polymers must be chosen in such a way that they are as large as is ary to obtain a
good phase separated morphology and good mechanical and thermal properties of the resulting
copolymer. The pre-polymer length must be low enough to be miscible with the chain-extender
at the polymerisation temperature. lly, this is achieved when Mn is 10 000 g/mol or less.
Generally, a hard segment content in the range of 10-90 wt.% based on total
weight of the multi-block copolymer, preferably of 25-90 wt.%, results in flexible, thermoplastic
materials with good degradation and swelling properties at the temperature of application
(viz. about 37 °C for medical applications).
In a further aspect the invention is directed to a process for preparing the phase
separated, thermoplastic multi-block copolymers of the invention, comprising a
chain-extension reaction of pre-polymer (A) and pre-polymer (B) in the presence of a
multifunctional chain-extender, thereby obtaining a randomly segmented multi-block
copolymer.
Segmented multi-block copolymers with structure (AB)r and (ABC)r can be made
by chain-extending a mixture of the pre-polymers, containing the hard and the soft segments
forming monomers of segments R1, H and R2 , and optionally R3, in the desired ratio with an
equivalent amount of a multifunctional chain-extender, ably an aliphatic molecule, more
preferably a diisocyanate such as 1,4-butanediisocyanate (BDI). The segmented copolymers of
structures (AB)r or (ABC)r are preferably made in solution. Suitably, the pre-polymer(s) are
dissolved in an inert organic solvent and the chain-extender is added pure or in solution. The
polymerisation temperature can be the same or even lower than the highest phase transition
temperature of the pre-polymers. Coupling reactions with dicyclohexyl iimide (DCC) are
preferably carried out in solution. Two (or three) pre-polymers that are all diol or diacid
terminated may be mixed in solution with a diacid or diol ated chain-extender,
respectively, after which DCC is added.
Polymerisation takes place for a time long enough to obtain an intrinsic viscosity
of the copolymer of 0.1 dl/g or higher (measured at 25 °C in form). The low
40 polymerisation temperature and short risation time will prevent from
transesterification so that the phase separated morphology is obtained and the monomer
distribution is the same as in the pre-polymers that build the copolymer. On the contrary, high
molecular weight random mers have to be prepared at higher temperatures (> 100 °C)
and for a much longer time to obtain a full incorporation of all the monomers. During that time
transesterification reactions will occur and a more random (i.e. less blocky) monomer
distribution is ed.
The materials obtained by chain-extending in the bulk can also be produced in
situ in an extruder.
If the chain-extender is a difunctional, aliphatic molecule and the pre-polymers
are linear, a linear copolymer is made; if one of the reactants (either the chain-extender or at
least one of the pre-polymers) or both have more than two functional , branched
structures may be obtained at sufficiently low conversion. The chain-extender can be a
difunctional aliphatic chain-extender, preferably a diisocyanate such as
1,4-butanediisocyanate.
The combination of crystalline and amorphous phase forming pre-polymers or
monomers is chosen in such a way to obtain a phase separated ted or block co-polyester
or polyester-carbonate with the ble degradation, swelling, physical and thermal
properties. Typically, the sic viscosity is larger than 0.1 dl/g and less than 10 dl/g
red at 25 °C in chloroform), preferably between 0.1-2 dl/g, and more preferably between
0.2-1 dl/g.
The multi-block segmented copolymers can be formed into formulations of various
shape and dimensions using any known technique such as, for example, emulsification
processes, ion, moulding, solvent casting, spray-drying, spray-freeze ,
electrospinning, or freeze drying. The latter technique is used to form porous materials.
Porosity can be tuned by addition of co-solvents, non-solvents and/or leachables. Copolymers
can be sed (either solid or porous) into microspheres, microparticles, nanospheres, rods,
films, sheets, sprays, tubes, membranes, meshes, fibres, plugs, coatings and other articles.
Products can be either solid, hollow or (micro)porous. A wide range of biomedical implants can
be manufactured for applications in for example wound care, skin ry, nerve
regeneration, vascular eses, drug delivery, meniscus reconstruction, tissue engineering,
coating of surgical s, ligament and tendon regeneration, dental and orthopaedic repair.
The copolymers can be used alone or can be blended and/or co-extruded with other absorbable
or sorbable polymers.
Furthermore, they can be used in pharmaceutical applications, e.g. for drug
delivery, e.g. in the form of microspheres, solid implants, gels, gs, films, sheets, sprays,
tubes, nes, meshes, fibres, plugs, and other configurations.
As will be illustrated in the examples below, the materials of the ion have
improved properties, including thermal, mechanical, processing compared to copolymers
described in the prior art.
Also described is a composition for the delivery of at least one biologically active
40 compound (e.g. a biologically active small molecule, protein or peptide) to a host, comprising
the at least one biologically active compound encapsulated in a matrix, wherein said matrix
comprises at least one phase separated, thermoplastic multi-block copolymer as defined
herein.
It was found that a biodegradable multi-block copolymer of the invention is
ularly suitable as delivery vehicle for a polypeptide, ng for the controlled release of
the polypeptide from the matrix into its environment, e.g. in the body of a subject.
The multi-block mers of the invention have many options for tuning the
e properties of the delivery ition for the specific application. The release rate of
the biologically active compound may for example be increased by:
• sing the molecular weight of the water-soluble polymer in pre-polymer (A) at constant
molecular weight of pre-polymer (A);
• increasing the molar ratio between pre-polymer (A) and pre-polymer (B);
• increasing the content of a monomer that gives a faster degrading polymer in pre-polymer
(A), e.g. by replacing e-caprolactone by D,L-lactide or glycolide or by replacing D,L-lactide
with glycolide;
• decreasing the molecular weight of pre-polymer (B) at a constant molar ratio between
pre-polymer (A) and pre-polymer (B) (this increases the pre-polymer (A) weight percentage
and also decreases the Tm of pre-polymer (B) and the total amount of crystalline phase
present);
• decreasing the molecular weight of pre-polymer (A) at a nt molecular weight of the
water-soluble polymer and molar ratio between pre-polymer (A) and pre-polymer (B);
and/or
• the use of an additional, third segment derived from a water-soluble r, whereby the
content of the water-soluble r is increased.
The release rate may be decreased by the opposite changes as mentioned above, as well as by
• increasing the Tm of segment B, e.g. by the use of a mixture of PLLA and PDLA as
pre-polymer (B) (instead of only PLLA) in a ratio such that complexation occurs
between PLLA and PDLA;
• the use of an additional, third segment derived from a water-soluble polymer diol, whereby
a diisocyanate is used as chain-extender and the water-soluble polymer content is held
constant or is decreased. The water-soluble polymer in the third segment is built in the
multi-block copolymer with a slowly degrading urethane bond, compared to a faster
degrading ester bond of the water-soluble polymer in pre-polymer (A).
Biologically active nds which may be contained in the multi-block
copolymer matrix, such as a poly(D,L-lactic acid)-co-PEG-co-poly(D,L-lactic acid)-b-PLLA
((PDLLA-co-PEG-co-PDLLA)-b-PLLA) matrix or a
poly(e-caprolactone)-co-PEG-co-poly(e-caprolactone)-b-PLLA co-PEG-co-PCL)-b-PLLA)
matrix, include but are not limited to ptide, non-protein small sized drugs having a
molecular weight which in general is 1000 Da or less and biologically active polypeptides.
Examples of non-peptide, non-protein small sized drugs which may be contained
40 in the polyetherester urethane matrix such as a (PDLLA-co-PEG-co-PDLLA)-b-PLLA matrix
or a PCL-co-PEG-co-PCL)-b-PLLA , include but are not d to anti-tumour agents,
icrobial agents, including antibiotics, cephalosporins, aminoglycosides; macrolides;
tetracyclines, chemotherapeutic agents including sulphonamides; urinary tract antiseptics;
drugs for anaerobic infections; drugs for tuberculosis; drugs for leprosy, antifungal agents,
antiviral agents, elminthiasis agents, anti-inflammatory, anti-gout agents, lly
acting (opoid) analgesics, local anaesthetics, drugs for Parkinson Disease, centrally acting
muscle relaxants, hormones and hormone antiagonists, corticosteroids, glucocorticosteroids,
androgens, androgenic steroids, anabolic steroids, antiandrogens, estrogens, estrogenic
steroids, anti-estrogens, tins; thyroid drugs and anti-thyroid drugs.
When a small-sized drug, such as those hereinabove bed, is ned in a
(PDLLA-co-PEG-co-PDLLA)-b-PLLA matrix, the PEG ent of the copolymer preferably
has a molecular weight of from 200 to 1500 g/mol, preferably from 600 to 1000 g/mol, and is
present in the copolymer in an amount of from 5 wt.% to 20 wt.% of the weight of the
copolymer, preferably in an amount of from 5 wt.% to 10 wt.% of the weight of the copolymer.
In general, the PLLA is present in the copolymer in an amount of from 20 wt.% to 90 wt.% of
the weight of the copolymer, ably in an amount of from 30 wt.% to 70 wt.% of the
copolymer. The at least one small-sized drug molecule may be present in the matrix in an
amount of from 0.1 wt.% to 80 wt.%, preferably from 1.0 wt.% to 40 wt.%, most preferably from
to 20 wt.%. If it is desired to increase the hydrophilicity of the multi-block copolymer, and
thereby increase the degradation rate of the copolymer and the release rate of the incorporated
biologically active compound, the copolymer may be modified by replacing partially or
completely the D,L-lactide of the hydrophilic segment by glycolide and/or by using a PEG
component with a higher molecular weight or by increasing the weight fraction of PEG
component in the pre-polymer segment. If it is desired to decrease the hydrophilicity of the
polymer, and thereby decrease the degradation rate of the copolymer, and the release rate of
the incorporated biologically active compound, the copolymer may be modified by replacing
partially or completely the D,L-lactide of the hydrophilic segment by olactone and/or by
using a PEG component with a lower molecular weight or by sing the weight fraction of
PEG component in the pre-polymer segment.
A polypeptide consists of amino acids linked by peptide bonds. Short polypeptides
are also referred to as es, whereas longer polypeptides are lly referred to as
proteins. One tion is that those polypeptide chains that are short enough to be made
synthetically from the constituent amino acids are called peptides rather than proteins.
However, with the advent of better synthetic techniques, polypeptides as long as hundreds of
amino acids can be made, including full ns like ubiquitin. Another convention places an
informal ng line at approximately 50 amino acids in length. This definition is at
arbitrary. Long polypeptides, such as the amyloid beta peptide linked to Alzheimer’s disease,
can be ered proteins; and small proteins, such as insulin, can be considered peptides. At
any rate, the skilled person will appreciate that essentially any type of polypeptide can be
ulated and subsequently released from a copolymer matrix.
40 In one embodiment, a composition of the ion comprises a biologically active
peptide or ically active protein. Encapsulated polypeptides preferably contain only
natural amino acids, gh non-natural amino acids (i.e. compounds that do not occur in
nature but that can be incorporated into a polypeptide chain) and/or amino acid analogues as
are known in the art may alternatively be employed. Also, one or more of the amino acids in a
polypeptide may be ed, for example, by the addition of a chemical entity such as a
carbohydrate group, a phosphate group, a farnesyl group, an nesyl group, a fatty acid
group, a linker for conjugation, onalisation, or other modification (e.g. alpha ion),
etc.
In a preferred ment, the modifications of the peptide lead to a more stable
peptide (e.g. greater half-life in vivo). These modifications may include ation of the
peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially
interfere with the desired biological activity of the e. In certain embodiments, the
modifications of the peptide lead to a more biologically active peptide.
The biologically active polypeptide is preferably selected from the group
consisting of protein/peptide drugs, enzymes, receptor ligands, neurotransmitters, inhibitory
peptides, tory peptides, activator peptides, cytokines, growth factors, monoclonal
antibodies, monoclonal antibodies fragments, anti-tumour peptides, antibiotics, antigens,
vaccines and hormones. Exemplary polypeptides to be encapsulated are mentioned in
US-A-5 980 948 and Crommelin et al., Int. J. Pharm. 2003, 266(1-2), 3-16. Of course, it is also
envisaged to encapsulate two or more distinct (biologically active) polypeptides.
The size of the polypeptide(s) can vary. In one embodiment, the polypeptide has
a molecular weight of 10 000 Da or less. It was found that polypeptides of such size are
particularly suitable to be encapsulated in the matrix of a copolymer comprising PEG as a
segment of lymer (A) and/or as an additional pre-polymer, said PEG having a number
average molecular weight of from 400 to 3000 g/mol, preferably from 600 to 1500 g/mol.
Alternatively, or in on, said PEG is present in an amount of from 5 wt.% to 60 wt.%
based on total weight of the copolymer, preferably of from 5 wt.% to 40 wt.%.
In another embodiment, said polypeptide is a ically active protein having
a molecular weight of 10 000 Da or more. These larger polypeptides are preferably
encapsulated in the matrix of a copolymer which ns PEG, as a segment of lymer
(A) and/or as an additional pre-polymer, and wherein said PEG has a number average
lar weight of from 600 to 5000 g/mol, preferably from 1000 to 3000 g/mol and/or
wherein said PEG is present in an amount of from 5 wt.% to 70 wt.% based on total weight of
the copolymer, more preferably of from 10 wt.% to 50 wt.%.
A composition of the invention can have any desirable appearance or shape. In one
embodiment, multi-block copolymers of the t invention are processed in the form of
microspheres, microparticles, sprays, an implant, a coating, a gel, a film, foil, sheet, membrane
or rod.
One specific aspect relates to a composition in the form of microspheres. In general
microspheres are fine spherical particles having a er of less than 1000 mm, and
40 containing a biologically active compound. The microsphere may be a homogeneous or
monolithic microsphere in which the biologically active compound is dissolved or dispersed
throughout the polymer matrix. It is also possible that the microsphere is of a reservoir type in
which the biologically active compound is surrounded by a polymer in the mononuclear or
polynuclear state. When the ically active compound is a small sized water-soluble drug,
the drug may first be dispersed in a hydrophobic or lipophilic excipient, which combination
then is dispersed in the form of particles, droplets, or microsuspensions in the polymer matrix.
Microspheres can then be formed from the on.
The microspheres may be prepared by techniques known to those skilled in the
art, including but not limited to coacervation, solvent tion/evaporation, spray drying or
spray-freeze drying techniques.
In one ment, the microspheres are prepared by a solvent
extraction/evaporation technique which comprises dissolving the multi-block copolymer in an
organic solvent such as dichloromethane, and emulsification of the multi-block copolymer
solution in an aqueous phase containing an emulsifying agent, such as polyvinyl alcohol (as
bed among others by Okada, Adv. Drug Del. Rev. 1997, 28(1), 43-70).
The characteristics, such as le size, porosity and drug loading of the
so-formed microspheres depend on the process parameters, such as viscosity or concentration
of the aqueous polyvinyl alcohol phase, concentration of the multi-block copolymer solution,
ratio of dichloromethane to s solution of , ratio of primary emulsion to polyvinyl
alcohol phase and the stirring rate.
When the microspheres are formed by a spray-drying process, a low tration
of multi-block copolymer from 0.5 wt.% to 5 wt.%, preferably about 2 wt.%, in the organic
solvent, such as dichloromethane, is employed. Spray-drying results in l in the
formation of porous, irregularly shaped particles.
As the microspheres are being formed, a biologically active compound is
encapsulated in the microspheres or microparticles. In general, when the solvent
extraction/evaporation technique is employed to encapsulate lipophilic compounds, the
compound is first dissolved in the solution of the multi-block copolymer in an c solvent
such as dichloromethane or ethyl acetate. The organic on is then subsequently fied
in an aqueous polyvinyl alcohol solution, which yields an -water (O/W) emulsion. The
organic solvent is then ted into the aqueous phase and evaporated to solidify the
microspheres.
In general, when the solvent ation que is employed to encapsulate
water-soluble compound, an aqueous solution of the compound is first emulsified in a solution
of the block copolymer in an organic solvent such as dichloromethane. This primary
emulsion is then subsequently emulsified in an aqueous polyvinyl l solution, which
yields a water-in-oil-in-water (W/O/W) emulsion. The organic solvent, such as dichloromethane
or ethyl acetate, is then extracted similarly to the O/W process route to solidify the
microspheres. Alternatively, water-soluble agents may be dispersed directly in a solution of
the multi-block copolymer in an organic solvent. The obtained dispersion is then uently
40 emulsified in an aqueous solution comprising a surfactant such as polyvinyl alcohol, which
yields a solid-in-oil-in-water (S/O/W) emulsion. The organic solvent is then extracted similarly
to the O/W process route to solidify the microspheres.
When W/O/W and S/O/W emulsification routes are used to ulate
water-soluble compound, it may be challenging to obtain microspheres with ient
ulation efficiency. Due to the water-soluble character of the compound, part of the
nd may be lost to the aqueous extraction medium such as aqueous polyvinyl alcohol
on. A viscosifier, such as gelatin, may be used in the al water phase, to decrease
diffusion of the compound in the internal water phase to the external water phase. Also,
additives may be added to the external water phase to decrease the solubility of the compound
in the external water phase. For this purpose, salts may be used or the pH may be adjusted.
Water-in-oil-in-oil ) or solid-in-oil-in-oil (S/O/O) emulsification routes
provide an interesting alternative to obtain microspheres with sufficient encapsulation
efficiency. In the W/O/O s the biologically active nd is, similar to a W/O/W
process, dissolved in an aqueous solution and emulsified with a solution of the polymer in an
organic solvent, such as lly dichloromethaneor ethyl e. Subsequently, a polymer
precipitant, such as silicon oil, is then slowly added under stirring to form embryonic
microparticles, which are then poured into heptane or hexane to extract the silicone oil and
organic solvent and solidify the microspheres. The microparticles may be collected by vacuum
filtration, rinsed with additional solvent and dried under vacuum. In the S/O/O emulsification
route the biologically active compound is, similar to a S/O/W process, dispersed as a solid
powder in a solution of the polymer in an organic solvent, such as dichloromethane or ethyl
acetate. uently, a polymer precipitant, such as n oil, is then slowly added under
ng to form embryonic microparticles, which are then poured into heptane or hexane to
extract the silicone oil and dichloromethane and solidify the microspheres.
Stabilising agents may be added to the aqueous solution of protein to prevent loss
of protein ty during processing into pheres. Examples of such stabilisng agents are
human serum albumin, gelatin and carbohydrates, such as trehalose, inulin and sucrose.
When the spray-drying technique is employed, an aqueous solution of the
compound is emulsified in a solution of the copolymer in an organic solvent such as methylene
chloride, as hereinabove described. The water-in-oil emulsion is then spray-dried using a spray
dryer.
In further embodiments, the composition of the invention is in the form of a
coating, an injectable gel, an implant (preferably an injectable implant) or a coated t.
The composition in the form of a coating may be d as a drug-eluting coating e.g. on a
medical implant, such as a vascular or y stent, an orthopaedic prosthesis or an ocular
implant.
ically active compounds may be formulated into injectable solid implants via
extrusion. Typically, the compound and multi-block copolymer powders are physically mixed
where after the resulting powder blend is introduced to the extruder, heated and processed to
40 yield formulations of the desired shape and dimensions, such as a small diameter cylindrical
rod. Instead of physical mixing of the compound and multi-block copolymer powders, the
compound and polymer may be co-dissolved in a suitable solvent or a sion of compound
in a solution of r in a suitable solvent may be prepared, followed by freeze-drying and
extrusion of the freeze-dried powder. The latter generally improves the blend neity and
the content uniformity of the implants.
Also described is a method of delivering a biologically active compound to a subject
in need thereof, comprising administering an effective dose of a composition as d herein
to said t.
The t is lly a mammal, preferably a human. However, veterinary use
of the invention is also encompassed. The method can have a therapeutic, prophylactic, and/or
cosmetic purpose. Any suitable mode of administration can be selected, depending on the
circumstances. For example, administering may comprise the parenteral, oral, arterial,
intra-articular, intra-venal, intra-ocular, epidural, intra-thecal, intra-muscular,
intra-peritoneal, intravenous, intra-vaginal, , topical or subcutaneous administration of
the composition. In one embodiment, the invention provides a method for delivering a
biologically active ptide of interest to a subject in need thereof, comprising
administering an effective dose of a composition according to the invention to said subject,
n the composition is in the form of microspheres, an injectable implant or an in situ
forming gel and n the composition is administered intra-ocularly, intra-arterially,
intra-muscularly or subcutaneously.
For topical stration, the microspheres may be contained in a gel, cream, or
ointment, and may, if desired, be covered by a barrier. Thus, the microspheres may n
one or more biologically active compounds employed in the treatment of skin diseases, such as
psoriasis, eczema, hoea, and dermatitis.
In another embodiment, the microspheres may be contained in a gel such as a
hyaluronic acid gel or a macromolecular polysaccharide gel. Such an embodiment is applicable
particularly to parenteral applications, such as during and after surgery.
When administered via injection, the microspheres may be contained in a
pharmaceutical carrier such as water, saline solution (for example, 0.9 %), or a solution
containing a surfactant in an amount of from 0.1 % w/v to 0.5 % w/v. Examples of surfactants
which may be ed include, but are not limited to, Tween 80 surfactant. The
ceutical carrier may further contain a ifier, such as sodium
carboxymethylcellulose.
When administered via injection, the microspheres have an average size of from 1
mm to 200 mm, preferably from 5 mm to 100 mm, most preferably from 10 mm to 50 mm. Such
microspheres, when administered in combination with an acceptable pharmaceutical carrier,
may be employed in the treatment of a variety of diseases or disorders, depending upon the
biologically active compound that is ulated. Thus, injectable formulations including the
microspheres of the invention may be employed in the treatment of systemic diseases such as
rheumatoid arthritis, hepatitis, diabetes, or metabolic syndromes, and locally confined
40 diseases such as osteoarthritis, renal diseases, mations, local pain processes, local
infections, local skin diseases, tumours (or their sites after surgical removal as a postoperative
treatment to y any tumour cells possibly remaining), prostate or breast cancer,
agromegaly, ocular diseases such as age-related macular degeneration, local brain diseases
(e.g. Parkinson’s disease), and cardiovascular diseases such as acute myocardial infarction,
chronic heart failure or arthrosclerosis. Such injectable formulations also may be employed in
long-term therapeutic treatments such as for example, ents with corticosteroids,
androgens, antiandrogens, estrogens, anti-estrogens, progestangenic agents, or thyroid
hormones, or with anti-tuberculosis, anti-leprosy, or anti-malaria drugs.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1A: DSC thermograms of 50LP10L20-LL40
Fig. 1B: DSC thermograms of 30LP30L40-LL40
Fig. 1C: DSC thermograms of 50CP10C20-LL40
Fig. 2: Cumulative release of lysozyme from films composed of 30LP10L20-LL40,
50LP10L20-LL40, 70LP10L20-LL40, C20-LL40 and 30CP30C40-LL40.
Films were loaded with 10 wt.% me. e was ed at 37 °C in
phosphate buffer pH 7.4 (n = 3).
Fig. 3: Cumulative release of bovine serum albumin (BSA) from films composed of
30LP10L20-LL40, 50LP10L20-LL40, 70LP10L20-LL40, 30LP30L40-LL40 and
30CP30C40-LL40. Films were loaded with 10 wt.% BSA. Release was measured at
37 °C in phosphate buffer pH 7.4 (n = 3).
Fig. 4: Effect of composition of the hydrophilic block of multi-block copolymers on
cumulative e of lysozyme from films. Films were composed of
50LP10L20-LL40, 50GP10C20-LL40 or 50CP10C20-LL40 (25 wt.% PEG1000) and
were loaded with 10 wt.% lysozyme. Release was measured at 37 °C in phosphate
buffer pH 7.4 (n = 3).
Fig. 5: Activity of lysozyme released from films ed of 30LP10L20-LL40 or
50LP10L20-LL40 containing 10 wt.% lysozyme (37 °C, phosphate buffer pH 7.4) and
lysozyme ty of lysozyme solutions (0.01 wt.%, phosphate buffer pH 7.4) stored
at 4 and 37 °C as a function of time (n = 3).
Fig. 6: In vitro release of BSA from microspheres composed of 30LP10L20-LL40 and
50CP10C20-LL40 loaded with 3-4 wt.% of BSA at 37 °C in ate buffer pH 7.4
(n = 3).
Fig. 7: In vitro release of IGF-1 from IGF-1 loaded 50CP30C40-LL40 and 30CP30C40-LL40
films made by solvent casting of W/O. Release was measured at 37 °C in phosphate
buffer pH 7.2 (n = 3). Solid lines ent IGF-1 release as measured by UPLC.
Dotted lines represent IGF-1 release as measured by ELISA.
Fig. 8: SEM photo of 0.2 wt.% IGF-1 loaded 50CP10C20-LL40 microspheres prepared via a
W/O/W double emulsion route.
Fig. 9: In vitro release of IGF-1 from 0.2 wt.% IGF-1 loaded microspheres prepared of
50CP10C20-LL40with different IVs. Release was measured at 37 °C in phosphate
buffer pH 7.2 (n = 3).
Fig. 10: SDS PAGE s of IGF-1 released from 50CP10C20-LL40 microspheres with 0.2
wt% IGF-1 target loading and prepared using various turrax speeds after 1
and 2 weeks.
Fig. 11: In vitro release Protein A (MW 15 000 Da) from films composed of
20LP10L20-LL40, 30LP6L20-LL40 and 30CP10C20-LL40 (Protein A content 5
wt.%; film thickness 80-120 mm). Release was measured at 37 °C in phosphate
buffer pH 7.4 (n = 3).
Fig. 12: SEM photo of 3-4 wt.% Protein A loaded microspheres composed of
30CP10C20-LL40 (IV 0.71 dl/g) ed using various amounts of inulin in the
inner aqueous phase A: 0 % inulin, B: 2 % inulin. 1: Overview. 2: Zoom-in.
Fig. 13: In vitro release of Protein A from microspheres composed of 30CP10C20-LL40 at
3-4 wt.% Protein A target loading with ally 2 or 5 wt.% of inulin coencapsulated
, at 37 °C in phosphate buffer pH 7.4 (n = 3).
Fig. 14: In vitro release of Protein A from microspheres composed of 30CP10C20-LL40 at
3-4 wt.% Protein A target loading and different polymer IV, at 37 °C in phosphate
buffer pH 7.4 (n = 3).
Fig. 15: SDS-PAGE results of Protein A ed from 30CP10C20-LL40 microspheres with
4 wt.% Protein A and 2 wt.% inulin target g after 1 (lane 4), 7 (lane 7), 14
(lane 8) and 21 (lane 9) days. Lane 5: Molecular weight markers. Lane 6: Protein A
standard. Note that the dark smears are due to ing of phosphate buffer salts.
Fig. 16: In vitro release of Peptide A (MW 2500) from films composed of 20LP10L20-LL40
(peptide load 5 and 10 wt.%; film thickness 80-100 mm) loaded. Release was
measured at 37 °C in phosphate buffer pH 7.4 (n = 3).
Fig. 17: In vitro release of Peptide A (MW 2500) from microspheres composed of
20LP10L20-LL40 (particle size 30 mm; peptide load 10 wt.%). Release was measured
at 37 °C in phosphate buffer pH 7.4 (n = 3).
Fig. 18 In vitro release of rapamycin from microspheres composed of various blends of
20LP1020-LL40-and 10LP10L20-LL40.
Fig. 19 SEM pictures of goserelin-loaded 20LP1020-LL40 microspheres prepared via the
W/O/O method
Fig. 20 In vitro e of goserelin from 20LP1020-LL40 microspheres prepared via the
W/O/O method
EXAMPLES
In the ing examples various biodegradable rystalline, phase separated
40 block copolymers were synthesised and evaluated for their processing and controlled
release characteristics. The polymers were composed of a lline L-lactide-based hard
segment B with a melting point (Tm) and a hydrophilic poly(ethylene glycol) (PEG)-based
segment A having a glass transition temperature (Tg) that was below body temperature under
physiological conditions. In the following examples PEG is denoted with its molecular weight
(MW). For example PEG1000 refers to PEG with MW 1000 g/mol.
EXAMPLE 1:
In this example, general procedures for the preparation of poly(DL-lactide-co-PEG)
prepolmer (A) are provided. rs were weighed into a three-necked bottle under nitrogen
atmosphere and dried at 50 °C in case of glycolide and D,L-lactide for at least 16 h under
reduced pressure. PEG was dried at 90 °C under reduced pressure for at least 16 h. PEG was
added to the monomer(s) under nitrogen atmosphere. Subsequently, stannous octoate was
added and the mixture was magnetically stirred and reacted at 140 °C for several days.
1H-NMR was med on a VXR Unity Plus NMR Machine n) operating at 300 MHz.
The d1 waiting time was set to 20 s, and the number of scans was 16. Spectra were recorded
from 0 to 14 ppm. Conversion and pre-polymer Mn was determined from 1H-NMR. 1H-NMR
samples were prepared by dissolving 10 mg of polymer into 1 ml of ated chloroform.
EXAMPLE 2:
This example describes the preparation of poly(DL-lactide-co-PEG1000) (pLP10L20)
with Mn 2000 g/mol. 149.84 grams (1.04 mol) of D,L-lactide (Purac) was weighed and 149.21 g
(0.149 mol) of PEG MW1000 (Ineos, PU grade) was added. 71.6 mg of stannous octoate (Sigma
Corp) was added (monomer/catalyst molar ratio = 5900) and the mixture was magnetically
stirred and reacted at 140 °C during 245 h. 1H-NMR showed 94.8 % monomer conversion. The
calculated lar weight (Mn) from in-weights was 2000 g/mol. Molecular weight as
determined by 1H-NMR was 1950 g/mol.
EXAMPLE 3
This example describes the preparation of poly(DL-lactide-co-PEG3000) (pLP30L40)
with Mn 4000 g/mol. 50.35 g (0.349 mol) of D,L-lactide (Purac) was weighed and 151.08 g (50.4
mmol) of PEG MW3000 (Sigma Corp) was added. 37.5 mg of stannous octoate (Sigma Corp)
was added (monomer/catalyst molar ratio = 4300) and the mixture was magnetically stirred
and d at 140 °C during 90 h. 1H-NMR showed 93.4 % r conversion. The
calculated lar weight (Mn) from ghts was 4000 g/mol. Molecular weight as
determined by 1H-NMR was 3940 g/mol.
EXAMPLE 4
This example describes the ation of poly(e-caprolactone-co-PEG1000)
pre-polymer (pCP10C20) with Mn 2000 g/mol. 100.81 g (0.101 mol) of PEG MW1000 (Ineos, PU
grade) was d into a three-necked bottle under nitrogen atmosphere and dried at 90 °C
40 for at least 16 h under reduced pressure. 101.76 g (0.892 mol) of e-caprolactone (Acros,
previously dried and distilled over CaH2 under reduced pressure) was added to the PEG under
nitrogen atmosphere and the e was heated to 135 °C. 57.9 mg of stannous octoate
(Sigma Corp) was added (monomer/catalyst molar ratio = 6200) and the mixture was
magnetically stirred and reacted at 135 °C during 76 h. 1H-NMR showed 100 % monomer
conversion. The calculated molecular weight (Mn) from in-weights was 2010 g/mol. Molecular
weight as determined by 1H-NMR was 1950 g/mol.
EXAMPLE 5
This e describes the preparation of poly(e-caprolactone-co-PEG3000)
pre-polymer (pCP30C40) with Mn 4000 g/mol. 176.60 g (58.9 mmol) of PEG MW3000 (Ineos,
PU grade) was weighed into a three-necked bottle under nitrogen atmosphere and dried at
90 °C for at least 16 h under reduced pressure. 59.4 g (0.520 mol) of e-caprolactone (Acros,
usly dried and led over CaH2 under reduced pressure) was added to the PEG under
nitrogen atmosphere and the mixture was heated to 135 °C. 69.6 mg of stannous octoate
(Sigma Corp) was added (monomer/catalyst molar ratio = 3000) and the mixture was
magnetically stirred and reacted at 135 °C during 243 h. 1H-NMR showed 100 % monomer
conversion. The calculated molecular weight (Mn) from in-weights was 2010 g/mol. Molecular
weight as determined by 1H-NMR was 1950 g/mol.
EXAMPLE 6
This example describes the preparation of poly(L-lactic acid) pre-polymer (LL4000)
with Mn = 4000 g/mol initiated by 1,4-butanediol (BDO). 399.89 g (2.77 mol) of L-lactide
(Purac) was weighed into a three-necked bottle under en here and dried at 50 °C
for at least 16 h under reduced pressure. 9.36 g (0.104 mol) of BDO (Acros, previously distilled
under reduced pressure) was added to the L-lactide under nitrogen atmosphere. 434 ml of
dioxane (Acros, previously dried and distilled over sodium wire) was added to dissolve the
L-lactide and BDO and the mixture was heated to 80 °C. 87.8 mg of stannous octoate (Sigma
Corp) was added (monomer/catalyst molar ratio = 12 800). The mixture was magnetically
stirred and reacted at 80 °C during 50.6 h. The polymer was retrieved from dioxane by
freeze-drying for 72 h to a final temperature of 50 °C. In case of polymer dissolved in dioxane,
the dioxane was first d under reduced pressure at 50 °C. 1H-NMR showed 96.5 %
r conversion. The calculated lar weight (Mn) from in-weights was 3940 g/mol.
Molecular weight as determined by 1H-NMR was 3900 g/mol. After freeze-drying dioxane
content was determined by 1H-NMR (300 MHZ, 50 mg of polymer dissolved into 1 ml of
deuterated chloroform, d1 = 30 s, 32 . 5 mg of dibromobenzene (Acros) was dissolved in
the sample for quantification of the e. Dioxane content was found to be 1193 ppm.
EXAMPLE 7
This e describes the general procedures used for the preparation of
multi-block copolymers. e-Caprolactone-co-PEG-co-e-caprolactone (CPC) or
40 D,L-lactide-co-PEG-co-D,L-lactide pre-polymers (LPL) (Mn 2000 g/mol) were heated to 50-80 °C
until they became more . The appropriate amounts of LL4000 pre-polymer (Mn 4000
g/mol) and CPC or LPL pre-polymer were ed into a glass ampoule supplied with
nitrogen inlet and dried at 50 °C for at least 48 h. Subsequently, the glass ampoule was
supplied with a mechanical stirrer. oxane (Acros, distilled over sodium) was added to a
polymer concentration of 30 wt.% and the contents of the ampoule were heated to 80 °C to
dissolve the pre-polymers. 0.900-0.990 equivalent (with respect to the pre-polymer hydroxyl
groups) of 1,4-butanediisocyanate (Bayer, distilled at reduced pressure) was added and the
reaction e was stirred mechanically for 16-22 h. Non-distilled dioxane was added to a
polymer tration of 20 wt.% to quench unreacted isocyanate groups. The reaction
mixture was further diluted with non-distilled dioxane to a polymer concentration of 10 wt.%.
The e was cooled to room ature, the reaction mixture was poured into a tray and
frozen at -18 °C. Subsequently, e was removed by placing the frozen on mixture
under vacuum at 30 °C. The polymer was stored in a sealed package at -18 °C. A small part of
the batch was analysed for thermal properties (mDSC), dioxane content (gas chromatography),
intrinsic viscosity and polymer composition (1H-NMR). Thermal analysis was performed by
Modulated Differential Scanning Calorimetry (mDSC). Samples of 5-10 mg were weighed in a
DSC pan. The measurement was performed on a DSC Q1000 (TA Instruments) using a
modulated temperature program. Amplitude was set to 1 °C, the modulation period to 60 s and
the heating rate to 5 °C/min. Samples were heated from -80 °C to 100-200 °C (depending on
the type of polymer). Intrinsic viscosity was ed using an Ubbelohde Viscosimeter (DIN),
type 0C, 0a or I, Schott Geräte supplied with a Schott AVS-450 Viscosimeter including a water
bath. The ements were performed in chloroform at room temperature. The polymer
tration in chloroform was such that the relative viscosity was in the range of 1.2 to 2.0.
Dioxane content was determined using a GC-FID headspace . Measurements were
performed on a GC-FID Combi Sampler supplied with an Agilent Column,
DB-624/30 m/0.53 mm. Samples were prepared in DMSO. Dioxane content was determined
using dioxane calibration standards.
EXAMPLE 8
This e describes the ation of
20(D,L-Lactide-co-PEG1000-co-D,L-lactide)2000-80(L-lactide)4000 (20LP10L20-LL40). 42.02 g of
LL40 pre-polymer (Mn 4040 g/mol, 10.40 mmol) and 10.16 g of
D,L-lactide-co-PEG1000-D,L-lactide pre-polymer (Mn 2000 g/mol, 5.08 mmol) were weighed and
ved in 100 ml of 1,4-dioxane at 80 °C. 1.8466 g (13.2 mmol) of 1,4-butanediisocyanate
was added (0.851 equivalent with respect to the pre-polymer hydroxyl groups) with 20 ml of
1,4-dioxane After 17 h the on was quenched with 88 ml of non-distilled dioxane and
further diluted with 255 ml of non-distilled dioxane. The e was removed by placing the
frozen reaction mixture under vacuum at 30 °C.
EXAMPLE 9
40 This example describes the preparation of
(D,L-lactide-co-PEG1000-co-D,L-lactide)2000-70(L-lactide)4000 (30LP10L20-LL40). 34.44 g of
LL40 pre-polymer (Mn 4020 g/mol, 8.57 mmol) and 14.95 g of D,L-lactide-co-PEG1000-D,L-lactide
pre-polymer (Mn 2040 g/mol, 7.33 mmol) were weighed and dissolved in 100 ml of 1,4-dioxane
at 80 °C. 2.7386 g (19.5 mmol) of 1,4-butanediisocyanate was added (1.231 equivalent with
respect to the pre-polymer hydroxyl groups) with 20 ml of 1,4-dioxane After 20 h the on
was quenched with 85 ml of non-distilled dioxane and further diluted with 240 ml of
non-distilled dioxane. The dioxane was removed by placing the frozen reaction mixture under
vacuum at 30 °C.
EXAMPLE 10
This example describes the preparation of
50(D,L-lactide-co-PEG1000-co-D,L-lactide)2000-50(L-lactide)4000 (50LP10L20-LL40). 19.59 g of
LL40 lymer (Mn 4060 g/mol, 4.83 mmol) and 19.57 g of D,L-lactide-co-PEG1000-D,L-lactide
pre-polymer (Mn 2040 g/mol, 9.59 mmol) were d and dissolved in 78 ml of 1,4-dioxane at
80 °C. 2.0018 g (14.3 mmol) of 1,4-butanediisocyanate was added (0.991 lent with
respect to the lymer hydroxyl groups) in 20 ml of 1,4-dioxane. After 20 h the reaction
was quenched with 67 ml of non-distilled dioxane and further diluted with 189 ml of
non-distilled dioxane. Dioxane was removed by placing the frozen reaction mixture under
vacuum at 30 °C.
EXAMPLE 11
This example describes the preparation of
70(D,L-lactide-co-PEG1000-co-D,L-lactide)2000-30(L-lactide)4000 (70LP10L20-LL40). 8.59 g of LL40
lymer (Mn 4020 g/mol, 2.14 mmol) and 19.96 g of D,L-lactide-co-PEG1000-D,L-lactide
lymer (Mn 2040 g/mol, 9.78 mmol) were weighed and dissolved in 48 ml of 1,4-dioxane at
80 °C. 1.648 g (11.8 mmol) of 1,4-butanediisocyanate was added (0.986 lent with respect
to the pre-polymer yl groups) with 20 ml of 1,4-dioxane After 21 h the reaction was
quenched with 49 ml of non-distilled dioxane, and further diluted with 147 ml of non-distilled
dioxane. The dioxane was removed by placing the frozen reaction mixture under vacuum at
°C.
EXAMPLE 12
This example describes the preparation of
(D,L-lactide-co-PEG3000-co-D,L-lactide)4000-70(L-lactide)4000 (30LP30L40-LL40). 29.96 g of
LL40 pre-polymer (Mn 4030 g/mol, 7.43 mmol) and 14.01 g of D,L-lactide-co-PEG1000-D,L-lactide
pre-polymer (Mn 4000 g/mol, 3.50 mmol) were weighed and dissolved in 83 ml of 1,4-dioxane at
80 °C. 1.52 g (10.8 mmol) of 1,4-butanediisocyanate was added (0.992 equivalent with respect
to the lymer hydroxyl groups) with 20 ml of 1,4-dioxane. After 21 h the reaction was
ed with 74 ml of non-distilled dioxane and further diluted with 222 ml of non-distilled
e. The dioxane was removed by placing the frozen reaction mixture under vacuum at
40 30 °C.
EXAMPLE 13
This example bes preparation of
50(e-caprolactone-co-PEG1000-co-e-caprolactone)2000-50(L-lactide)4000 (50CP10C20-LL40). 24.34 g
of LL40 pre-polymer (Mn 4030 g/mol, 6.04 mmol) and 23.87 g of
e-caprolactone-co-PEG1000-e-caprolactone pre-polymer (Mn 2010 g/mol, 11.9 mmol) were
weighed and dissolved in 95 ml of 1,4-dioxane at 80 °C. 2.4098 g (17.2 mmol) of
tanediisocyanate was added (0.960 equivalent with respect to the pre-polymer hydroxyl
groups) with 20 ml of 1,4-dioxane. After 18 h the reaction was quenched with 82 ml of
non-distilled dioxane and further diluted with 246 ml of stilled e. The dioxane
was removed by placing the frozen reaction mixture under vacuum at 30 °C.
EXAMPLE 14
This example describes preparation of
(e-caprolactone-co-PEG3000-co-e-caprolactone)4000-70(L-lactide)4000 (30CP30C40-LL40). 35.84 g
of LL40 pre-polymer (Mn 4030 g/mol, 8.89 mmol) and 14.79 g of
e-caprolactone-co-PEG3000-e-caprolactone pre-polymer (Mn 4010 g/mol, 3.69 mmol) were
weighed and dissolved in 100 ml of 1,4-dioxane at 80 °C. 1.7428 g (12.4 mmol) of
1,4-butanediisocyanate was added (0.988 equivalent with respect to the pre-polymer yl
groups) with 20 ml of 1,4-dioxane. After 18 h the reaction was quenched with 83 ml of
non-distilled dioxane and further diluted with 240 ml of non-distilled dioxane. The e
was removed by placing the frozen reaction mixture under vacuum at 30 °C.
EXAMPLE 15
The synthesised multi-block copolymers were analysed for chemical composition,
molecular weight and residual dioxane content. Table 1 shows the collected analysis results for
20LP10L20-LL40, L20-LL40, L20-LL40, 70LP10L20-LL40, 30LP30L40-LL40,
50CP10C20-LL40, 30CP30C40-LL40. The actual composition of the copolymers, as determined
by 1H-NMR from L/P and C/P molar ratios resembled the target composition well. All polymers
had an intrinsic viscosity between 0.7 and 1.1 dl/g. Dioxane contents were well below 1000
ppm indicating effective removal of dioxane by -drying.
The multi-block copolymers were analysed for their thermal properties to confirm
their phase separated morphology. Results are shown in Table 2. Figure 1 shows typical DSC
thermograms of 50LP10L20-LL40 (Figure 1A), 30LP30L40-LL40 (Figure 1B) and
C20-LL40 e 1C) multi-block copolymers. All multi-block copolymers exhibited a
melting temperature (Tm) at approximately 120-133 °C, due to melting of the LL40 segment.
As expected, the melting enthalpy (DHm) of the crystalline LLA40 t increased with
increasing amount of the segment. 70LP10L40-LL40, 50CP10C20-LL40 also ted a Tm at
approximately 85 °C, which is attributed to g of less perfect crystals of LL40.
Copolymers containing PEG3000 showed a Tm at approximately 40 °C, due to melting of the
PEG. The glass transition temperature (Tg) of the block copolymers is in general in
between that of pre-polymer (A) and pre-polymer (B), indicating phase mixing of the
amorphous pre-polymer (A) with the amorphous content of pre-polymer (B). The Tg of
LP10L20-LL40 type multi-block copolymers increased from -18 to 50 °C when sing the
LLA40 segment from 30 to 80 wt.%. The Tg of these multi-block copolymers is in between that
of pre-polymer (A) (pLP10L20, Tg -37 °C) and lymer (B) (LL40, Tg ~ 50 °C) and is thus
attributed to mixing of amorphous polylactide of the semi-crystalline LL40 block and PEG.
50CP10C20-LL40 had a Tg of -48 °C, which is rly attributed to mixing of amorphous
PEG, polycaprolactone and polylactide. Table 3 shows the swelling degree of the block
copolymers. To measure the ng characteristics of polymers, polymer films were made by
pouring a 13 wt.% polymer solution in dichloromethane (approximately 300 mg of polymer
with 1.5 ml of dichloromethane), on a glass plate and spreading the polymer solution with a
g knife or poured into a Teflon™ mould. The dichloromethane was left to evaporate
slowly overnight and the residual dichloromethane was removed by vacuum drying at 20 °C.
Resulting films had a thickness of 100-200 mm. For the swelling tests, 15-40 mg of circular
films with a diameter of imately 25 mm were weighed and immersed in a flask
ning 10 ml of phosphate buffer pH 7.4 (ISO-15814). The s were stored in an oven
at 37 °C. At each sampling time point, samples were collected and excess buffer solution was
removed from the surface where after the samples were weighed on a 4-decimal balance. All
tests were performed in duplicate. The swelling degree was found to increase gradually with
the content of PEG of the copolymers and with PEG MW at approximately constant PEG
content.
Table 1: ted results regarding the chemical composition, intrinsic viscosity and residual dioxane content of multi-block copolym ers
20LP10L20-LL40, 30LP10L20-LL40, 50LP10L20-LL40, 70LP10L20-LL40, 30LP30L40-LL40, C20-LL40, 30CP30C40-LL40.
20LP10L20-LL40 30LP10L20-LL40 50LP10L20-LL40 70LP10L20-LL40 30LP30L40-LL40 C20-LL40 30CP30C40-LL40
Molar L/P ratio
126.1 78.2 42.1 26.3 137.4 27.8 130.1
in-weights
Molar L/P ratio
128.5 75.9 42.6 25.7 129.9 26.8 131.8
1H-NMR
Molar C/P ratio
- - - - - 8.8 7.8
in-weights
Molar C/P ratio
- - - - - 8.2 8.8
1H-NMR
Intrinsic viscosity
0.73 0.85 0.89 0.70 0.79 1.05 0.69
(dl/g)
Dioxane content
<200 256 <200 <200 <200 <200 <200
(ppm)
Table 2: Thermal characteristics of multi-block copolymers (MBCP) 20LP10L20-LL40, 30LP10L20-LL40, 50LP10L20-LL40,
70LP10L20-LL40, 30LP30L40-LL40, 50CP10C20-LL40, 30CP30C40-LL40 and their pre-polymers (PP) A and B.
20LP10L20-LL40 L20-LL40 50LP10L20-LL40 70LP10L20-LL40 30LP30L40-LL40 50CP10C20-LL40 30CP30C40-LL40
Tg (°C) MBCP 50 5 -15 -18 - -48 -
Tm (°C) MBCP 134 126 123 85/120 37/132 87/126 43/133
DHm (J/g) MBCP 50 39 31 2/4 1/40 4/13 35/25
Tg (°C) PP A -37 -37 -37 -37 -39 -67 -67
Tm (°C) PP A - - - - 35/42 43 43
DHm (J/g) PP A - - - - 37 (both peaks) 91 85
Tg (°C) PP B 43 46 48 46 57 57 57
Tm (°C) PP B 85/131 117/134 136 4 137 137 137
DHm (J/g) PP B 24 (both peaks) 28 (both peaks) 32 28 (both peaks) 57 57 57
Table 3: Composition and swelling of multi-block mers 20LP10L20-LL40, 30LP10L20-LL40, 50LP10L20-LL40, 70LP10L20-LL40,
30LP30L40-LL40, 50CP10C20-LL40, 30CP30C40-LL40.
wt.% Segment A wt.% Segment B MW PEG wt.% PEG ng degree (-)
20LP10L20-LL40 20 80 1000 10 xx
30LP10L20-LL40 30 70 1000 15 1.03
50LP10L20-LL40 50 50 1000 25 1.13
70LP10L20-LL40 70 30 1000 35 1.26
30LP30L40-LL40 30 70 3000 22.5 1.16
50CP10C20-LL40 50 50 1000 25 1.18
30CP30C40-LL40 30 70 3000 22.5 1.67
EXAMPLE 16
In this example various hydrophilic phase separated multi-block copolymers
described in the examples above were evaluated for their n release teristics using
bovine serum albumin (BSA, 69 kDa) and lysozyme (14 kDa) as model proteins.
Protein-loaded films containing 10 wt.% protein were prepared by mixing of
approximately 150 ml of 20 wt.% protein solution with 1.5 ml of dichloromethane containing
300 mg of polymer for 30 s with an Ultra turrax at 18 000 rpm. The emulsion was spread on a
glass plate with a casting knife or poured into a Teflon™ mould. The dichloromethane was left
to evaporate slowly overnight and the residual dichloromethane was removed by vacuum
drying at 20 °C. Resulting films had a thickness of 80-120 mm.
For the e tests, 20 mg of protein loaded film were weighed and immersed in
vials containing 5 ml of phosphate buffer pH 7.4 and stored in an oven at 37 °C. At each
sampling point, 1 ml of release medium was sampled and ed with 1 ml of fresh buffer.
The n content of the release samples was determined with a Bicinchoninic Acid (BCA)
assay (Pierce) using an Easys Expert 96 well plate reader.
The biological activity of released me was measured by means of a bacteria
lysis test. Lysozyme loaded films were prepared as described above. A 0.01 wt.% lysozyme
solutions was prepared to serve as a control by weighing 2.1 mg of lysozyme and adding 20 ml
of phosphate buffer. Lysozyme-loaded films were weighed and immersed in vials containing 5
ml of phosphate buffer pH 7.4. Vials containing lysozyme-loaded films as well as y
prepared lysozyme solutions were stored in an oven at 37 °C. At each sampling point, 1 ml of
release medium was sampled and replaced with 1 ml of fresh buffer. The protein content of the
release samples was determined by BCA as described above. The activity of (released)
me was determined by following the change in turbidity at 450 nm for 3 min of a
bacteria sion (Micrococcus lysodeikticus, Sigma, 0.21 mg/ml) to which 10 ml of sample
was added. A UV-VIS spectrometer (Varian) was used for this purpose. Samples were diluted
if necessary to obtain a lysozyme concentration of 5-100 mg/ml. The lysozyme activity of the
samples was calculated by comparing the slope of the obtained curves (the slope relates to the
lysozyme activity) with the slope of a curve obtained with a fresh me on.
Figure 2 and 3 show the release of respectively lysozyme and bovine serum
albumin from the films. The results show that by changing the PEG content and PEG MW the
release rate and profile can be varied. Lysozyme was released over periods varying from a few
days up to approximately 3 months. Due to its larger size the release rate of BSA was lower
resulting in release over periods g from a few days up to approximately 4 months.
Furthermore, the release of lysozyme could be tuned by introducing different (combinations of)
monomers adjacent to the PEG group in the hydrophilic block of the multi-block copolymers.
The resulting block copolymers 0L20-LL40, 50GP10C20-LL40 and
50CP10C20-LL40) ned 25 wt.% PEG1000 and ted similar swelling degrees, but
ent degradation rates leading to various release profiles for the encapsulated lysozyme.
(Figure 4).
Figure 5 shows the activity of lysozyme released from 10 wt.% lysozyme-loaded
films of 30LP10L20-LL40 or L20-LL40 (phosphate buffer pH 7.4, 37 °C). As a l
the activity of lysozyme of the 0.01 wt.% lysozyme solutions 4 stored at 4 or 37 °C was
measured (phosphate buffer pH 7.4). The results show that lysozyme released from the films
over a period of imately one month retained its biological activity, indicating that the
structural integrity and biological activity of lysozyme was not only preserved during the
encapsulation process but also during long-term presence of lysozyme in the hydrated and
swollen polymer matrix at 37 °C prior to release.
EXAMPLE 17
In this example 30LP10L20-LL40 (IV 0.85 dl/g) and 50CP10C20-LL40 (IV 1.06
dl/g) type phase separated copolymers were used to formulate BSA into microspheres.
BSA loaded microspheres were prepared of C20-LL40 (IV 1.06 dl/g) and
30LP10L20-LL40 (IV 0.85 dl/g) hydrophilic phase separated multi-block copolymers by a
solvent evaporation method using procedures as disclosed by by Kissel et al., J. Contr. Rel.
1996, 39(2), 315-326 and Meinel et al., J. Contr. Rel. 2001, 70(1-2), 193-202. BSA (25-50 mg of)
was dissolved in about 150 mg of ultra-pure water and emulsified with 2-3 ml of a solution of
50CP10C20-LL40 (15 % w/v) or 30LP10L20-LL40 (23 % w/v) in dichloromethane for 60 s using
an Ultra turrax IKA T18 operated at 20 000 rpm yielding a in-oil (W/O) emulsion). The
so-obtained primary emulsion was then emulsified in about 80-130 ml of UP-water containing
4.0 wt.% PVA for 30 s using an Ultra turrax IKA T18 ed at 14 000 rpm yielding a
water-in-oil-in water (W/O/W) emulsion. The so-obtained secondary emulsion was gently
stirred for 2 h at 600 rpm at room temperature. Due to the ation of the
dichloromethane, the polymer precipitated from the solution to yield pheres. After 3 h
(the time ary to achieve almost complete evaporation of the dichloromethane) the
formed microspheres were collected by centrifugation, and the microspheres were washed
three times with 100-200 ml of an aqueous solution of 0.05 wt.% Tween 20 in ultra-pure water.
Finally, the microspheres were lyophilised.
For IVR tests, 2 ml of 100 mM phosphate buffer (pH 7.4, 0.02 wt.% NaN3) in case
of 30LP10L20-LL40 microspheres and 25 mM NaPi buffer (pH 7.2, 105 mM NaCl, 0.01 wt.%
Tween 80, 0.02 wt.% NaN3) in case of 50CP10C20-LL40 microspheres was added to 20 mg of
microspheres. The sample was incubated at 37 °C and at each sampling point 1.8 ml of sample
was taken and refreshed with release buffer. BSA content was measured with BCA protein
assay in case of 30LP10L20-LL40 microspheres and with UPLC (eluent A: 1 wt.% TFA in
UP-water, eluent B: 0.085 wt.% TFA in acetonitrile, 95/5 v/v A/B to 5/95 A/B in 25 min) in case
of 50CP10C20-LL40 microspheres.
The le size distribution of the pheres was measured by Coulter
counter. Approximately 1 mg of microspheres were dispersed in 50-100 ml of Isotron II
solution by gently stirring and the particle size was measured with a Coulter counter ed
with a 100 mm measurement cell.
The BSA content of the microspheres was ined by dissolving 5-10 mg of
microspheres, accurately weighted, in 5.0 ml of acetonitrile. After fugation, 4 ml of
supernatant was removed and 5 ml of PBS was added. BSA content was measured with UPLC
t A: 0.1 wt.% TFA in UP-water, eluent B: 0.1 wt.% TFA in acetonitrile, 90/10 v/v A/B to
/90 v/v A/B in 4 min).
Table 4 lists the particle size, encapsulation efficiency (EE) of the BSA loaded
microspheres prepared. Figure 6 shows the in vitro release of BSA from 30LP10L20-LL40
microspheres with 5 wt.% BSA target loading and 50CP10C20-LL40 microspheres with 10
wt.% BSA target loading. BSA was released from 30LP10L20-LL40 microspheres for almost 3
months in a linear fashion without significant burst. 50CP10C20-LL40 microspheres release
BSA for almost ~ 3 months in a linear fashion without significant burst, where after slower
release followed for another ~ 1.5 months.
Table 4: Average particle size, BSA content and encapsulation efficiency of
BSA loaded 50CP10C20-LL40 and 30LP10L20-LL40
microspheres.
Polymer grade Average size Content EE (%)
(mm) (wt.%)
50CP10C20-LL40 14 2.8 33
30LP10L20-LL40 18 4.3 85
EXAMPLE 18
In this example various hydrophilic phase separated multi-block mers
prepared as described in the examples above were used to prepare Insulin-like Growth
Factor I (IGF-1) loaded film and microsphere formulations.
IGF-1 loaded films were prepared by dissolution of 0.18 g of polymer into 1.46 g of
dichloromethane and subsequent emulsification by Ultra ing with IGF-1 dissolved in
ultra pure water at 18 000 rpm for 30 s or by using ultrasound at 100 W for 5 s. The emulsion
was poured into a Teflon™ mould. Dichloromethane was left to ate overnight and
residual dichloromethane was removed by vacuum drying overnight. 20 mg films were cut and
put on e at 37 °C with 1 ml of phosphate buffered saline (PBS, 25 M pH 7.2, 105 mM
NaCl, 0.01 wt.% Tween 80 and 0.02 wt.% NaN3). At predetermined time points, samples were
taken and the sampled amount was refreshed by fresh buffer.
IGF-1 loaded microspheres were ed by a solvent extraction/evaporation
based W/O/W emulsification process. 2.78 mg of IGF-1 and 51.8 mg of BSA were dissolved in
143 ml of UP-water in an Eppendorf cup and fied in a solution of 0.47 g of
50CP10C20-LL40 (IV 1.05 dl/g) in 2.62 g of dichloromethane using an Ultra turrax (20 000
rpm, 60 s). The so-obtained primary on was then emulsified in 81 ml of UP-water
containing 4.0 wt.% PVA using an Ultra turrax (14 000 rpm for 60 s), and d for 2 h at 600
rpm at room temperature. The resulting microspheres were collected on a 5 mm membrane
filter and washed with 1 l of UP-water containing 0.05 wt.% Tween 80. Finally, the
microspheres were lyophilised.
Approximately 1 mg of microspheres were dispersed in 50-100 ml of Isotron II
solution by gently ng and the particle size was measured with a Coulter counter ed
with a 100 mm measurement cell.
The IGF-1 and BSA content were determined by dissolving 5 mg of microspheres,
accurately weighted, in 0.3 ml of acetonitrile. Subsequently, 1.2 ml of PBS was added and
gently shaken. After centrifugation, the IGF-1 and BSA content in the supernatant were
determined by UPLC. Procedure was performed in triplicate.
Using a cial ch ELISA (R&D Systems), the concentration of human
insulin-like Growth Factor I ) in a sample was ed to confirm that
ncapsulated and released IGF-1 was still capable to bind with the capture and detection
antibody after release and thus no protein degradation at that level has occurred. The e
and detection antibody of the kit were specific for natural and recombinant IGF-1 and as a
standard recombinant IGF-1.
To investigate the structural integrity of released IGF-1, 100-300 ng of IGF-1
collected from release samples was denaturated using Laemli/b-mercapto-ethanol buffer and
loaded on an ‘any KD TGX’ pre-cast mini gel and separated under denaturating conditions at
100-200 V using 1 × Tris/ Glycine/ SDS as separating buffer, and stained overnight in colloidal
CBB staining agent. A Dual Xtra Protein marker (Bio-Rad) was used to determine the n
size of the separated proteins.
Figure 7 shows the in vitro release of IGF-1 from 50CP30C40-LL40 and
30CP30C40-LL40 polymer films loaded with 0.6 wt.% of IGF-1 as measured by UPLC and
ELISA. IGF-1 was released from the 50CP30C40-LL40 films in about 7 days whereas IGF-1
was slowly released from the 30CP30C40-LL40 polymer films with a cumulative release of
about 40 % after 28 days. Since the cumulative release of IGF-1 as measured by UPLC was
nearly identical to the tive release of IGF-1 as measured by ELISA it was concluded
that the released IGF-1 was structurally intact and ically active.
Microspheres with 0.5 wt.% of IGF-1 target loadings were prepared of
50CP10C20-LL40 with IV 1.05 and 0.68 dl/g by a double emulsification process. The
microspheres had a smooth e (Figure 8) and encapsulation encies varying between
40 and 60 %. The volume average particle size (d50) as measured with a Coulter counter
equipped with a 100 mm measurement cell was 54.4 mm with a CV (coefficient of variation) of
61 %. Figure 9 shows IGF-1 release from these microspheres in vitro. Complete release of
IGF-1 within 2 days was obtained for microspheres composed of 50CP10C20-LL40 with IV
0.68 dl/g. IGF-1 release from microspheres composed of 50CP10C20-LL40 with IV 1.05 dl/g
was slower with te release achieved after approximately 6 days. Released IGF-1 was
structurally intact as could be concluded from the SDS-PAGE s (Figure 10), which did
not show any degradation nor aggregation of the protein.
EXAMPLE 19
In this example various hydrophilic phase separated multi-block copolymers
(20LP10L20-LL40 (IV 0.58 dl/g), 30LP6L20-LL40 (IV 0.60 dl/g) and 30CP10C20-LL40 (IV 0.71
dl/g)) prepared as described in the examples above were used to prepare film formulations
loaded with a highly water-soluble biologically active polypeptide with a molecular weight of
kDa (Protein A). Furthermore, 30CP10C20-LL40 multi-block copolymers with various IV
(0.81, 0.71 and 0.65 dl/g) were used to formulate Protein A into microspheres formulations.
n A loaded films were prepared by a solvent casting method. 10 mg of
Protein A was dissolved in 123 mg of UP-water and emulsified in a on of 0.18 g of
polymer in 1.46 g of romethane using an Ultra turrax (18 000 rpm, 60 s). The
so-obtained primary emulsion was poured in a Teflon™ mould and the dichloromethane was
evaporated ght. al dichloromethane was removed by vacuum drying.
n A loaded microspheres were ed by a solvent extraction/evaporation
based W/O/W emulsification process. 21 mg of Protein A (5 wt.% target loading) was dissolved
in 156 ml of er optionally containing inulin in an Eppendorf cup and emulsified in a
solution of 0.4 g of polymer in 2.1 g of dichloromethane using an Ultra turrax (20 000 rpm,
60 s). The so-obtained primary emulsion was then emulsified in 70 ml of UP-water containing
4.0 wt.% PVA using an ultraturrax (14 000 rpm for 60 s), and stirred for 2 h at 600 rpm at
room temperature. The resulting microspheres were collected on a 5 mm membrane filter and
washed with three times 100 ml of UP-water containing 0.05 wt.% Tween 80. Finally, the
microspheres were lyophilised.
Approximately 10 mg of microspheres were dispersed in 50-100 ml of Isotron II
solution by gently stirring and the particle size was measured with a Coulter r equipped
with a 100 mm ement cell.
The Protein A content was determined by dissolving 5 mg of microspheres,
accurately weighted, in 0.3 ml of acetonitrile. After fugation, the atant was
removed and the residual ACN was evaporated. 1.95 ml of PBS was added. Protein A content
was measured with UPLC (eluent A: 0.1 wt.% TFA in UP-water, eluent B: 0.1 wt.% TFA in
acetonitrile, 80/20 v/v A/B to 10/90 A/B in 3 min).
For SEM imaging, a small amount of microspheres was adhered to carbon
conductive tape and coated with gold for 3 min. The sample was imaged using a 10 kV electron
beam.
The in vitro release kinetics of Protein A-loaded films and microspheres were
measured in 100 mM of phosphate buffer pH 7.4 (20 mg of film in 2 ml). The samples were
incubated at 37 °C. At each sampling point, 1.8 ml of sample was taken and refreshed with 1.8
ml of phosphate buffer. Protein A content was measured with UPLC (eluent A: 0.1 wt.% TFA
in er, eluent B: 0.1 wt.% TFA in acetonitrile, 80/20 v/v A/B to 10/90 A/B in 3 min).
SDS-PAGE was performed in reducing mode with 4-20 % Tris-HCl gels. Per slot
ml of protein solution was applied for samples and Protein A rd. For the marker, 2 ml
was applied to the slot. The amount of protein added per slot was either 75 or 150 ng. Samples
were prepared by dilution with 12 mM PBS pH 7.4 or er to a Protein A tration of
either 150 or 300 ng/20 ml. Subsequently, Laemmli working solution (Laemmli buffer
containing 1 % of mercaptoethanol) was added in ratio 1:1 v/v. The samples were heated to
~ 90 °C for 5 min and d to the gels. The gels were clamped in the electrophoresis cell and
running buffer (Tris/Glycine/SDS pH 8.3) was added. The s and standards were applied
to the gels, and the gels were run for 15 min at 100 kV. The voltage was subsequently set to
200 kV and the gels were run until a good separation of the lar weight standards was
obtained. The gels were washed with UP-water and d with silver reagent.
Figure 11 shows the in vitro release of Protein A from 20LP10L20-LL40 (10 wt.%
of PEG MW 1000), 30LP6L20-LL40 (9 wt.% of PEG MW 600) and C20-LL40 (15 wt.%
of PEG MW 1000). C20-LL40-based films ed Protein A relatively fast with a
cumulative release of Protein A of 100 % after 3 months. By replacing PEG1000 by PEG600,
which leads to reduction of the swelling degree, the release of Protein A could be slowed down
and near first-order diffusion controlled release kinetics were obtained leading to a tive
release of ~ 75 % after 4 months. Reduction of the release rate of Protein A could also be
achieved by lowering the weight fraction of the hydrophilic LP10L20 block in the polymer. By
using 20LP10L20-LL40 (10 wt.% PEG1000) release could be further slowed down, and after an
initial small burst of less than 15 %, well-controlled release kinetics of Protein A were obtained
with cumulative release of ~ 65 % in 6 months. The data y show that Protein A release
kinetics can be controlled by the choice of polymer.
Protein-A-loaded microspheres were prepared of 30CP10C20-LL40 loaded with 3-4
wt.% of Protein A. Optionally, 2 or 5 wt.% of inulin was co-encapsulated to enhance the release
rate of Protein A. The effect of r molecular weight on protein release kinetics was
studied by studying the release kinetics of n A from microspheres composed of
30CP10C20-LL40 polymers with different intrinsic viscosity. For all Protein A loaded
microspheres, spherical pheres were obtained. For microspheres with co-encapsulated
inulin, the surface ty increased with increasing inulin content, as shown on the SEM
pictures in Figure 12. Table 5 lists the particle size and encapsulation efficiency (EE) of
Protein A of the microspheres. Figure 13 shows that after a small initial burst, Protein A was
released at a constant rate. It was observed that the burst decreased and linearity increased
with decreasing inulin content. Without inulin present ~ 70 % was released in 3 months, while
90-100 % was released when 2 or 5 wt.% inulin was co-encapsulated. Release of Protein A from
30CP10C20-LL40 films containing 2 or 5 wt.% of co-encapsulated inulin were similar. Release
data is shown up to ~ 4 months. Expected duration of release for Protein A from
C20-LL40 microspheres is approximately 6 months.
Figure 14 shows the release kinetics of Protein A from 30CP10C20-LL40 films
with different intrinsic viscosity (IV) of the polymer. The release rate of n A increased
with increasing polymer IV. For 30CP10C20-LL40 polymers with an IV 0.71 or 0.81 dl/g,
sustained release of Protein A was obtained with cumulative release of 60-70 % after 2
months. The release kinetics of Protein A from microspheres composed of 30CP10C20-LL40
with an IV of 0.58 dl/g were significantly different. The initial release rate up to one month
was significantly lower, but Protein A release accelerated between 1 and 3 months, where after
it slowed down again, giving a total duration of release of approximately 5 months. The data
clearly show that Protein A can be released from microspheres in linear fashion for at least 4
months and that release cs can be controlled by the co-encapsulation of sugars, such as
inulin, as well as by the intrinsic viscosity of the polymer.
The structural ity of Protein A released from microspheres was studied by
SDS-PAGE. SDS-PAGE confirmed that Protein A released for at least 21 days consisted
mainly of native Protein (Figure 15). These results show that 30CP10C20-LL40 microspheres
provide a suitable matrix for the long-term e of structurally intact n A.
Table 5: Overview of Protein A loaded microspheres characteristics with 3-4 wt.%
n A target loading.
MSP # r IV apsulated Particle Protein A EE Protein A
(dl/g) inulin (wt.%) size content (wt.%) (%)
(mm)
#1 0.71 0 52 3.7 100
#2 0.71 2 57 3.3 90
#3 0.71 5 55 1.8 54
#4 0.57 0 33 4.0 100
#5 0.81 0 43 0.7 24
EXAMPLE 20
In this example the hydrophilic phase separated multi-block copolymer
20LP10L20-LL40 (IV 0.73 dl/g) prepared as described in the examples above was used to
prepare film and microsphere formulations loaded with a biologically active polypeptide with a
molecular weight of 2.5 kDa (Peptide A).
Peptide A loaded films were prepared by a solvent casting method. 10 (for 5 wt.%
loading) or 20 mg (for 10 wt.% g) of Peptide A was ved in 123 mg of er and
emulsified in a solution of 0.18 g of 20LP10L20-LL40 (IV 0.76 dl/g) in 1.46 g of
dichloromethane using an Ultra turrax (18 000 rpm, 30 s). The so-obtained primary emulsion
was poured in a Teflon mould and the dichloromethane was evaporated overnight. Residual
dichloromethane was removed by vacuum drying.
Peptide A loaded pheres were prepared by a solvent evaporation based
double emulsion s. 50 mg of Peptide A was dissolved in PBS and emulsified in a solution
of 0.5 g of L20-LL40 (IV 0.73 dl/g) in 2 g of dichloromethane using an Ultra turrax
(24 000 rpm, 60 s). The so-obtained primary emulsion was then emulsified in 200 ml of
UP-water ning 4.0 wt.% polyvinyl l using an ultraturrax (14 000 rpm for 30 s), and
stirred for 3 h at 600 rpm at room temperature. The resulting microspheres were centrifuged,
the supernatant was removed and the microspheres were washed three times with 200 ml of
UP-water containing 0.05 wt.% Tween 20. Finally, the microspheres were lyophilised. The
particle size distribution was ed with a Coulter Counter. Approximately 10 mg of
microspheres were dispersed in 50-100 ml of Isotron II solution by gently stirring and the
le size was measured with a 100 mm measurement cell.
Peptide A content of microspheres was ined by dissolving 5-10 mg of
microspheres, accurately weighted, in 5.0 ml of acetonitrile. After centrifugation, 4 ml of
supernatant was removed and 5 ml of PBS was added. Peptide A content was measured with
HPLC (eluent A: 1 wt.% TFA in UP-water, eluent B: 0.085 wt.% TFA in acetonitrile, 95/5 v/v
A/B to 5/95 A/B in 25 min).
The in vitro release cs of Peptide A from films and microspheres were
measured in PBS pH at 37 °C. Peptide A containing films or microspheres (5-20 mg) were
weighed into a vial and 2 ml of PBS was added. The vials were ted at 37 °C and sampled
at pre-determined time-points. At each sampling point 75-90 % of release medium was
collected and replaced by fresh PBS. Peptide A content of e samples was determined
with HPLC (eluent A: 1 wt.% TFA in UP-water, eluent B: 0.085 wt.% TFA in acetonitrile, 95/5
v/v A/B to 5/95 A/B in 25 min).
Figure 16 shows the in vitro release of Peptide A from 20LP10L20-LL40 films.
Peptide A was released from 5 wt.% loaded 20LP10L20-LL40 films in a linear fashion for at
least 5 months without significant burst. For 20LP10L20-LL40 films with a higher Peptide A
loading (10 wt.%), burst release increased to 15 %. After approximately 2 months, release was
similar to the 5 wt.% loaded films.
Peptide A loaded 20LP10L20-LL40 pheres had an average particle size of
mm and a Peptide A content of 10.3 wt.%, representing an encapsulation efficiency of 100 %.
Figure 17 shows that Peptide A MSP exhibited a low burst release of approximately 10 wt.%
followed by zero-order release kinetics for at least 40 days.
EXAMPLE 21
In this example, hydrophilic phase separated multi-block copolymers
20LP10L20-LL40 (Example 8) and 10LP10L20-LL40 were used to prepare microspheres
loaded with rapamycin (MW 914 Da). The hylene glycol component of the polymers had
a lar weight of 1000 g/mol.
Rapamycin loaded microspheres with a target load of 20 wt.% rapamycin were
prepared by a t evaporation method using a single oil-in-water (O/W) emulsion route.
The polymers were dissolved in various blend ratios in dichloromethane to a concentration of
about 20 wt.%, and the required amount of rapamycin was added. The r/rapamycin
solution was then emulsified in 200 ml of UP-water containing 4.0 wt.% polyvinyl alcohol
(PVA) using an Ultra turrax (14 000 rpm for 30 s), and then stirred with a magnetic stirrer for
3 h at 300 rpm at room temperature. The microsphere dispersion was concentrated by
centrifugation and the microspheres were washed three times with 50 ml of aqueous 0.05 wt.%
Tween 20 solution. Finally, the microspheres were lyophilised.
The particle size distribution was measured with a Coulter Counter.
Approximately 10 mg of microspheres were dispersed in 50-100 ml of Isotron II on by
gently ng and the particle size was measured with a 100 mm ement cell.
Rapamycin content of microspheres was determined by dissolving 5-10 mg of
microspheres, accurately weighted, in 5.0 ml of acetonitrile. After fugation, 4 ml of
supernatant was removed and 5 ml of PBS was added. Rapamycin content was measured with
HPLC (eluens: acetonitrile/water 70/30 v/v; 278 nm).
The in vitro e kinetics of rapamycin from microspheres were measured at
37 °C in 10 mM PBS pH 7.4 containing 0.5 wt.% SDS rapamycin containing microspheres
(5-20 mg) were weighed into a vial and 2 ml of release medium was added. The vials were
incubated at 37 °C and d at pre-determined time-points. At each sampling point 75-
90 % of release medium was collected and replaced by fresh PBS. Rapamycin content of e
samples was determined with HPLC.
The pared cin microspheres had an average size of 35 mm and a
rapamycin content varying from 17 to 20 wt.%, representing encapsulation efficiencies of 89 %
to 100 %. Figure 18 shows the release of rapamycin from microspheres composed of various
blends of 20LP10L20-LL40 and 10LP10L20-LL40. Rapamycin release from
20LP10L20-LL40-based microspheres was vely fast, whereas release of rapamycin from
10LP10L20-LL40-based microspheres was very slow. By blending the two polymers
microspheres with intermediate release profiles were obtained.
EXAMPLE 22
In this example, goserelin acetate loaded microspheres were prepared of the
hydrophilic phase separated block copolymer 20LP10L20-LL40 by means of a
water-in-oil-in-oil process. 62.6 mg of goserelin acetate was dissolved in 150 ml of UP-water
(29.4 wt.%) and emulsified with a solution of 0.5 g of -LLA40 polymer in 7.4 g of
dichloromethane in a scintillation vial (Ultra turrax, 20 000 rpm, 60 s). 13.5 g of the polymer
precipitant (silicon oil, 350 cSt) was then slowly added (2-5 min) under nt stirring
(12 000 rpm) to form embryonic microparticles. The embryonic microparticles were then
poured into 550 ml of heptane at room temperature (13.5:1 ratio of dichloromethane to
heptane solvent). The extraction vessel was closed to prevent excessive evaporation of the
extraction . After approximately 3 h of extraction, the microparticles were collected by
vacuum filtration, rinsed with additional heptane and dried under . The microspheres
had an average size of 67 mm and a goserelin t of 8.3 %, representing an encapsulation
efficiency of 88 %.
The particle size bution was measured with a Coulter Counter.
Approximately 10 mg of microspheres were dispersed in 50-100 ml of n II solution by
gently stirring and the particle size was measured with a 100 mm measurement cell.
Goserelin t of microspheres was determined by dissolving 5-10 mg of
microspheres, accurately weighted, in 5.0 ml of acetonitrile. After centrifugation, 4 ml of
supernatant was removed and 5 ml of PBS was added. Goserelin content was measured with
HPLC (eluens: water / acetonitrile / trifluoracetic acid 72/28/0.1, 220 nm).
The in vitro release kinetics of goserelin from microspheres were measured in PBS
(192 mM pH 7.4 containing 0.01 % tween 80 and 0.02 % sodium azide) at 37 °C. lin
containing microspheres (5-20 mg) were d into a vial and 2 ml of release medium was
added. The vials were incubated at 37 °C and sampled at termined oints. At each
sampling point 75-90 % of release medium was collected and replaced by fresh PBS. Goserelin
content of release samples was determined with HPLC.
The so-prepared goserelin-loaded 20LP10-LLa40 microspheres had a spherical
and smooth appearance (Figure 19), an average size of 71 mm (CV 47 %) and a goserelin
content of 8.3 % representing an encapsulation efficiency of 88 %. Figure 20 shows the release
of goserelin from the microspheres.
EXAMPLE 23
In this example, lysozyme-loaded microspheres were prepared of the hydrophilic
phase separated multi-block copolymer 30CP10L20-LL40 by means of a solid-in-oil-in-oil
process ). 0.43 g of 30CP10L20-LL40 was dissolved in 7.4g of dichloromethane in a
scintillation vial (5.4 wt.%), and 0.074 g of dried inulin-stabilized lysozyme
microparticles (lysozyme/inulin ratio: 1:2 w/w) with a particle size of 1-2 mm were added to the
polymer on, and the dispersion was nised by Ultra turrax (20 000 rpm, 60 s).
11.46 g of the polymer precipitant (silicon oil, 350 cSt) was then slowly added (2-5 min) under
constant stirring (12 000 rpm) to form nic microparticles. The embryonic microparticles
were then poured into 550 ml of heptane at room temperature (13.5:1 ratio of dichloromethane
to heptane solvent). The extraction vessel was closed to prevent excessive evaporation of the
extraction medium. After imately 3 h of extraction, the articles were collected by
vacuum filtration, rinsed with additional heptanes and dried by vacuum filtration. The
microspheres had an average size of 59 mm and a lysozyme content of 4.1-5.6 %, representing
an encapsulation efficiency of 80-100 %.
In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that
such documents, or such sources of information, in any iction, are prior art, or form part
of the common general knowledge in the art.
In the description in this specification reference may be made to subject matter
that is not within the scope of the claims of the current ation. That subject matter should
be readily identifiable by a person skilled in the art and may assist in putting into practice the
invention as defined in the claims of this application.
Claims (69)
1. Biodegradable, semi-crystalline, phase separated, thermoplastic block copolymer, the copolymer being characterised in that: a) it comprises at least one hydrolysable pre-polymer (A) segment and at least one hydrolysable pre-polymer (B) segment, b) said multi-block copolymer having a Tg of 37 °C or less and a Tm of 110-250 °C under physiological conditions; c) the segments are linked by a multifunctional chain-extender; d) the segments are randomly distributed over the polymer chain; e) at least part of the pre-polymer (A) segment is derived from a water-soluble polymer.
2. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, having a structure according to formula (1): ‒[R1-H-R1-Q1-R4-Q2]x‒[R2-Q3-R4-Q4]y‒[R3-Q5-R4-Q6]z‒ (1) wherein R1 is selected from the group consisting of amorphous ter, amorphous polyetherester or amorphous polycarbonate; or an amorphous pre-polymer that is obtained from combined ester, ether and/or carbonate ; H is derived from a water-soluble polymer selected from the group consisting of polyethers, polyvinylalcohol; polyvinylpyrrolidone; polyvinylcaprolactam, poly(hydroxyethylmethacrylate), polyphosphazenes, polyorthoesters, polyorthoesteramides or copolymers of the previous polymers; R2 is ed from the group consisting of crystalline or semi-crystalline polyester, polyetherester, polycarbonate or polyanhydride; or lymers of ed ester, ether, anhydride and/or carbonate groups; variable z is zero or a positive integer; variables x and y are both a positive r; R3 is optionally present and is derived from a soluble polymer that is chosen from the group consisting of polyethers, polyvinylalcohol, polyvinylpyrrolidone, polyvinylcaprolactam, poly(hydroxymethylmethacrylate), polyphosphazenes, thoesters, polyorthoesteramides or copolymers of the previous polymers; and R4 is derived from the chain-extender and consists of an aliphatic C2-C8 alkylene group, optionally substituted by a C1-C10 ne, the tic group being linear or , the C1- C10 alkylene side group ally containing protected S, N, P or O moieties; Q1-Q6 are each independently selected from amine, urethane, amide, carbonate, ester and anhydride.
3. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 1, wherein said extender is a difunctional aliphatic chain-extender.
4. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer ing to claim 3, wherein the extender is a diisocyanate.
5. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer ing to claim 4 wherein the diisocyanate is 1,4-butane diisocyanate.
6. Biodegradable, semi-crystalline, phase separated, plastic multi-block mer according to any one of claims 1-5, wherein pre-polymer (A) comprises reaction products of cyclic monomers and/or non cyclic monomers, wherein said non cyclic monomers are selected from the group consisting of ic acid, ic acid, adipic acid, sebacic acid, lactic acid, glycolic acid, hydroxybutyric acid, ethylene glycol, diethylene glycol, 1,4-butanediol and/or 1,6-hexanediol, and wherein said cyclic monomers are selected from the group consisting of glycolide, lactide, e-caprolactone, d-valerolactone, trimethylene carbonate, tetramethylenecarbonate, oxepaneone, 1,4-dioxaneone (para-dioxanone) and/or cyclic anhydrides such as oxepane-2,7-dione.
7. radable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to any one of claims 1-6, wherein said water-soluble polymer is ed from the group consisting of polyethers , polyvinylalcohol (PVA) polyvinylpyrrolidone (PVP), polyvinylcaprolactam, poly(hydroxyethylmethacrylate) (poly-(HEMA)), polyphosphazenes, polyorthoesters, polyorthoesteramides or copolymers of the previous polymers.
8. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 7, wherein the polyethers are ed from polyethylene glycol (PEG), tramethyleneoxide (PTMO) and polypropyleneglycol (PPG).
9. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 7, wherein said water-soluble polymer is d from poly(ethylene glycol) (PEG) having a Mn of 150-5000 g/mol.
10. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to any one of claims 1-9, wherein a water-soluble polymer is present as an additional pre-polymer.
11. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer ing to any one of claims 1-10, wherein said pre-polymer (B) segment comprises a crystallisable polymer derived from hydroxyalkanoate, glycolide, L-lactide or D- lactide.
12. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 11, wherein said pre-polymer (B) segment comprises L-lactide pre-polymers and ide pre-polymers in such s and ratio that stereocomplexation between L-lactide and D-lactide is achieved.
13. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block mer according to claim 11, wherein said pre-polymer (B) is poly(L-lactic acid) with an Mn of 1000 g/mol or more.
14. radable, rystalline, phase separated, thermoplastic multi-block copolymer according to claim 11, wherein said pre-polymer (B) is poly(L-lactic acid) with an Mn of 2000 g/mol or more.
15. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 11, wherein said lymer (B) is poly(L-lactic acid) with an Mn of 3000 g/mol or more.
16. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to any one of claims 1-14, wherein the swelling ratio under physiological conditions varies from 1 to 4.
17. Biodegradable, rystalline, phase separated, plastic multi-block mer according to claim 16, wherein the swelling ratio under physiological conditions varies from 1 to 2.
18. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 16, wherein the swelling ratio under physiological conditions varies from 1 to 1.5
19. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to any one of claims 1-18, wherein said mer has an intrinsic viscosity of at least 0.1 dl/g.
20. Biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to claim 19, wherein said copolymer has an intrinsic viscosity of between 0.2 dl/g and 2 dl/g.
21. Process for preparing a biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to any one of claims 1-20, comprising i) ming a chain extension reaction of pre-polymer (A) and pre-polymer (B) in the presence of a unctional chain-extender, wherein pre-polymer (A) and (B) are both diol or diacid terminated and the chain-extender is di-carboxylic acid or diol terminated; ii) performing a chain extension reaction using a coupling agent, wherein pre-polymer (A) and (B) are both diol or diacid terminated and the ng agent is dicyclohexyl carbodiimide.
22. A biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer prepared by a method of claim 21.
23. Use of a biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to any one of claims 1-20 and 22 in the cture of a medicament.
24. Use according to claim 23, wherein the medicament is in the form of pheres, microparticles, nanoparticles, nanospheres, rods, implants, gels, coatings, films, sheets, , tubes, membranes, meshes, fibres, or plugs.
25. A composition for the delivery of at least one biologically active compound to a host, comprising at least one biologically active compound encapsulated in a matrix, wherein said matrix comprises at least one biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer ing to any one of claims 1-20 and 22.
26. A composition ing to claim 25, wherein said at least one biologically active nd is a ptide non-protein small sized drug, or a biologically active polypeptide.
27. A composition according to claim 26, wherein said non-peptide, non-protein small sized drug comprises one or more selected from the group consisting of an anti-tumour agent, an anti-microbial agent, a sephalosporin, an aminoglycoside, a macrolide, a tetracycline, a chemotherapeutic agent, a urinary tract antiseptic, a drug for anaerobic infections, a drug for tuberculosis, a drug for leprosy, an antifungal agent, an antiviral agent, an anti-helminthiasis agent, an nflammatory agent, an anti-gout agent, a lly acting ) analgesic, a local anaesthetic, a drug for Parkinson’s disease, a centrally acting muscle relaxant, a hormone or hormone anti-agonist, a corticosteroid, a glucocorticosteroid, an androgen, an androgenic steroid, an anabolic steroid, an anti-androgen, an estrogen, an enic steroid, an anti-estrogen, a progestin, a thyroid drug and an anti-thyroid drug.
28. A ition according to claim 26, wherein said biologically active polypeptide ses one or more selected from the group consisting of a protein/peptide drug, an enzyme, a receptor ligand, a neurotransmitter, an inhibitory peptide, a regulatory peptide, an activator peptide, a cytokine, a growth factor, a monoclonal antibody, a monoclonal antibody fragment, an anti-tumour peptide, an antibiotic, an antigen, a vaccine, and a hormone.
29. A composition according to any one of claims 25-28, wherein said biologically active compound is a non-peptide, non-protein small molecule having an Mn which is 1000 Da or less, as a segment of pre-polymer (A) and/or as an additional pre-polymer, wherein said water e polymer i) has a molecular weight of from 200 to 1500 g/mol; and/or ii) is present in an amount of from 5 wt.% to 20 wt.%.
30. A composition according to claim 29, wherein said water soluble polymer is poly(ethylene ).
31. A composition according to claim 30, wherein said poly(ethylene glycol) has a molecular weight of 600 to 1000 g/mol.
32. A composition according to claim 30 or 31, wherein said poly(ethylene glycol) is present in an amount of from 5 wt.% to 10 wt.%.
33. A composition according to any one of claims 25, 26 or 28, wherein said biologically active compound is a biologically active polypeptide having a molecular weight which is 10 000 Da or less, as a segment of pre-polymer (A) and/or as an additional pre-polymer, and wherein said water soluble polymer i) has a molecular weight of from 400 to 3000 g/mol; and/or ii) is present in an amount of from 5 wt.% to 60 wt.%.
34. A composition according to claim 33, wherein said water soluble polymer is poly(ethylene glycol).
35. A composition according to claim 34, n said poly(ethylene glycol) has a molecular weight of from 600 to 1500 g/mol.
36. A composition according to claim 34 or 35, wherein said poly(ethylene glycol) is present in an amount of from 5 wt.% to 40 wt.%.
37. A composition according to any one of claims 25, 26 or 28, wherein said ically active compound is a biologically active polypeptide having a lar weight of 10 000 Da or more, as a segment of pre-polymer (A) and/or as an additional pre-polymer, and wherein said water soluble polymer i) has a lar weight of from 600 to 5000 g/mol; and/or ii) is present in an amount of from 5 wt.% to 70 wt.%.
38. A composition according to claim 37, wherein said water soluble r is poly(ethylene glycol).
39. A composition according to claim 38, wherein said poly(ethylene glycol) has a lar weight of from 1000 to 3000 g/mol.
40. A composition according to claim 38 or 39, wherein said poly(ethylene glycol) is present in an amount of from 10 wt.% to 50 wt.%.
41. A composition according to any one of claims 25-40, in the form of pheres, microparticles, rticles, nanospheres, rods, ts, gels, coatings, films, , sprays, tubes, membranes, meshes, fibres, or plugs.
42. A composition according to any one of claims 25-41 in the form of microspheres and/or microparticles, wherein the average diameter of the microspheres and/or microparticles is in the range of 0.1-1000 mm.
43. A ition according to claim 42, wherein microspheres and/or microparticles are in the range of 1-100 mm.
44. A composition according to claim 42, wherein microspheres and/or microparticles are in the range of 10-50 mm.
45. A composition according to any one of claims 42-44, n the biologically active compound is dissolved or dispersed throughout the polymer matrix.
46. A composition according to any one of claims 42-44, wherein the microsphere comprises a reservoir wherein biologically active compound is contained, surrounded by a polymer in mononuclear or polynuclear state.
47. A composition according to any one of claims 25-46 for treating rheumatoid arthritis, hepatitis, es, metabolic syndromes, osteoarthritis, renal disease, inflammation, local pain processes, local infections, local skin diseases, s (or their sites after surgical removal as a postoperative treatment to destroy any tumour cells possibly remaining), prostate or breast cancer, agromegaly, ocular diseases such as age-related macular ration, local brain diseases such as Parkinson’s disease, and vascular diseases such as acute myocardial infarction, chronic heart failure or arthrosclerosis.
48. A use of a composition according to any one of claims 25-47, in the manufacture of a medicament for treating rheumatoid arthritis, hepatitis, diabetes, metabolic syndromes, osteoarthritis, renal disease, inflammation, local pain processes, local infections, local skin diseases, tumours (or their sites after surgical removal as a postoperative treatment to destroy any tumour cells possibly ing), prostate or breast cancer, agromegaly, ocular diseases, local brain diseases, and cardiovascular diseases.
49. A use as claimed in claim 48, n the ocular diseases is age-related macular degeneration.
50. A use as claimed in claim 48, wherein the local brain diseases is Parkinson’s disease.
51. A use as claimed in claim 48, wherein the cardiovascular diseases is acute myocardial infarction, chronic heart failure or arthrosclerosis.
52. A method of manufacturing a composition according to any one of claims 42-46, comprising the successive steps of a) emulsifying an aqueous solution of a water-soluble biologically active compound in a on of a biodegradable, semi-crystalline, phase separated, thermoplastic block copolymer according to any one of claims 1-20 and 22 in an organic t; b) subsequently emulsifying the ant emulsion of a) in an aqueous solution comprising a surfactant, thereby yielding a water-in-oil-in-water (W/O/W) emulsion; and c) extracting the organic solvent to solidify microspheres.
53. A method as claimed in claim 52, wherein the organic t is dichloromethane or ethyl acetate.
54. A method as claimed in claim 52, wherein the surfactant is polyvinyl alcohol.
55. A method of manufacturing a composition ing to any one of claims 42-46, sing the successive steps of a) dispersing the biologically active compound as a solid powder in a solution of a biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer ing to any one of claims 1-20 and 22 in an organic solvent; b) emulsifying the resultant dispersion of a) in an aqueous solution comprising a surfactant, thereby yielding a solid-in-oil-in-water (S/O/W) emulsion; and c) extracting the organic solvent to fy the microspheres.
56. A method ing to claim 55, wherein the organic solvent is dichloromethane or ethyl acetate.
57. A method according to claim 55, wherein the surfactant is polyvinyl alcohol.
58. A method of manufacturing a composition according to any one of claims 42-46, comprising the successive steps of a) emulsifying an aqueous solution of a water-soluble biologically active compound in a solution of a biodegradable, semi-crystalline, phase ted, thermoplastic multi-block copolymer according to any one of claims 1-20 and 22 in an c solvent; b) adding a polymer precipitant, to the resultant emulsion of a) to form embryonic microparticles; and c) extracting the polymer precipitant and the organic solvent to fy the microspheres.
59. A method according to claim 58, n the c solvent is dichloromethane or ethyl acetate.
60. A method according to claim 58, wherein the polymer precipitant is silicon oil.
61. A method of manufacturing a composition according to any one of claims 42-46, comprising the successive steps of a) dispersing the biologically active compound as a solid powder in a solution of a radable, semi-crystalline, phase separated, thermoplastic multi-block copolymer according to any one of claims 1-20 and 22 in an organic solvent; b) adding a polymer precipitant, to the resultant dispersion of a) to form embryonic microparticles; and c) extracting the polymer itant and the organic solvent to fy the microspheres.
62. A method according to claim 61, wherein the c solvent is dichloromethane or ethyl acetate.
63. A method according to claim 61, wherein the polymer precipitant is silicon oil.
64. A composition ed by the method of any one of claims 49-63.
65. A biodegradable, semi-crystalline, phase separated, thermoplastic multi-block copolymer as claimed in claims 1 or 22, substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.
66. A process as claimed in claim 21, substantially as herein described with reference to any example f and with or without reference to the accompanying drawings.
67. A use as claimed in claims 23 or 48, ntially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.
68. A composition as claimed in claims 25 or 64, substantially as herein described with reference to any example thereof and with or without reference to the anying drawings.
69. A method as claimed in claims 52, 55, 58 and 61, substantially as herein described with nce to any example thereof and with or without reference to the accompanying drawings.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11174987.5 | 2011-07-22 | ||
EP11174987 | 2011-07-22 | ||
PCT/NL2012/050529 WO2013015685A1 (en) | 2011-07-22 | 2012-07-23 | Biodegradable, semi-crystalline, phase separated, thermoplastic multi block copolymers for controlled release of biologically active compounds |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ620392A NZ620392A (en) | 2016-02-26 |
NZ620392B2 true NZ620392B2 (en) | 2016-05-27 |
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