Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DESCRIPTION
BIODEGRADABLE BIO-ABSORBABLE MATERIAL FOR CLINICAL PRACTICE
AND METHOD FOR PRODUCING THE SAME
Technical Field
The present invention relates to a biodegradable
bio-absorbable material for clinical practice, which can be
used for a medical device made of biodegradable bio-absorbable
material, such as suture thread, vascular stmt, biological
cell carrier , and carriers of drug and the like , and a method
for producing the same.
Background of the Invention
Bio-absorbable polymers for use as medical materials such
as vascular stent and suture thread include for example
polylactic acid, polyglycolic acid, a copolymer of the two,
namely polyglactin, polydioxanone, and polyglyconate (the
copolymer of trimethylene carbonate and glycolide).
Such bio-absorbable polymers are degraded and absorbed
in biological organisms. Therefore, such bio-absorbable
polymers are widely used. Because the dynamic properties
thereof such as tensile strength and the degradation rate thereof
for absorption are individually nearly definite, the
bio-absorbable polymers turn fragile when the dynamic
properties are enhanced, involving the reduction of the
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degradation rate. When the degradation rate is increased,
alternatively, the dynamic propertiesare deteriorated. Thus,
disadvantageously, the bio-absorbable polymers have only
limited purposes for use and are applied to limited sites.
Disclosure of the Invention
The present invention relates to a biodegradable
bio-absorbable material for clinical practice, which is a
copolymer produced by copolymerizing a bio-absorbable monomer
and a cyclic depsipeptide so that the content of the depsipeptide
can adjust the dynamic properties and degradation rate thereof ,
without any occurrence of inflammation and other problems.
The amount of the depsipeptide to be included is at about 2
to 60mo1 % . Below 2mol ~ , the effect thereof cannot be exerted.
At 60mo1 0 or more, the resulting dynamic properties are too
much deteriorated. Many types of bio-absorbable polymers can
be utilized. Depending on the type of a bio-absorbable polymer
or the amount of a bio-absorbable copolymer to be blended, the
amount of the depsipeptide to be included outside the limit
range of the amount of the depsipeptide to be included as
described above may sometimes involve the exertion of the effect .
Therefore, the ratio of the amount thereof to be included is
not a definite value.
Brief Description of the Drawings
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Fig. 1 depicts the structure view of depsipeptide; Fig.
2 depicts the structure view of a terpolymer with a depsipeptide
unit; Fig.3 depicts the explanatory scheme of the synthesis
of the depsipeptide; Fig. 4 depicts the structure view of the
terpolymer of a ring-opened and copolymerized depsipeptide;
Fig . 5 depicts the chart of the 1H-NMR spectrum of the terpolymer;
Fig. 6 depicts the graphs of the hydrolysis tests with a
degradation solution containing a buffer alone; Fig. 7 depicts
the results of the enzymatic degradation profiles of the
terpolymers and each of the homopolymers with proteinase K;
Fig. 8 depicts the degradation profiles of the copolymers with
the depsipeptides; Fig. 9 shows the relations among the amounts
of the depsipeptides and the degradation rates; Fig. 10 is a
figure or table showing the synthetic conditions of each of
the copolymers and homopolymers, the yields and molecular
weights of the polymers; Fig. 11 is a figure or table depicting
the thermal properties of each of the copolymers and the
homopolymers; Fig. 12 is a figure or table depicting the
mechanical properties (tensile strength) of the terpolymers
and the thermal properties thereof ; Fig . 13 is a figure or table
depicting the changes of various physico-chemical properties
of the terpolymers before and after degradation with proteinase
K; and Fig. 14 is a figure or table depicting the relations
between the amounts of depsipeptides and the thermal properties .
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Best Mode for Carrying out the Invention
So as to describe the invention in more detail , the
invention is now described with reference to the attached
drawings.
The structure of the depsipeptide is shown in Fig. 1.
As shown in the figure, the R group in a side chain is
an alkyl group such as methyl group, isopropyl group and isobutyl
group, while the R' group in a side chain is an alkyl group
such as methyl group and ethyl group.
Concerning examples of the depsipeptide, depsipeptides are
synthesized from amino acid and a hydroxy acid derivative , using
chloroacetyl chloride, 2-bromopropionyl bromide and
2-bromo-n-butyryl bromide asthe hydroxy acid derivative, which
are L-MMO, L-DMO, and L-MEMO, in the order of the above
derivatives . All of them are applicable to the invention. The
enzymatic degradation level of a copolymer from such
depsipeptide monomer and a bio-absorbable monomer
f -caprolactone ( CL ) with proteinase K is in the order of L-MMO/CL
> L-DMO/CL > L-MEMO/CL.
As to the depsipeptide synthesized from amino acid and a hydroxy
acid derivative, amino acids such as L-alanine, L-(DL- or
D-)valine, and L-leucine are used to prepare depsipeptides,
namely DMO, PMO and BMO in the order of the above amino acids .
All of them are applicable to the invention. The enzymatic
degradation level of a copolymer from such depsipeptide monomer
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and a bio-absorbable monomer e-caprolactone (CL) with
proteinase K is in the order of DMO/CL > PMO/CL > BMO/CL . The
enzymatic degradation level thereof with cholesterol esterase
is in the order of PMO/CL > BMO/CL z DMO/CL.
First embodiment
A terpolymer was prepared by copolymerizing a cyclic
depsipeptide (DMO), L-lactide (L-LA) and t.-caprolactone.
Fig . 2 is the structure view of the copolymer of a cyclic
depsipeptide with CL. U expresses depsipeptide unit.
At first, 3,6-dimethyl-2,5-morpholine-dione (DMO) was
synthetically prepared as a cyclic depsipeptide. The cyclic
depsipeptide is a cyclic ester amide prepared from a-amino acid
and a u-hydroxy acid derivative. Herein, DL-alanine and
DL-2-bromopropionyl bromide were used as a-amino acid and
cx-hydroxy acid derivative, respectively.
At the first step of the synthesis , the Schotten-Baumann
reaction between alanine and 2-bromopropionyl bromide was
carried out in an aqueous alkaline solution, for peptide linking
to prepare 2-bromopropionyl alanine (Fig. 3).
In other words, 150 ml of an aqueous solution of DL-alanine
(53.4 g; 0.6 mol) in 4N NaOH (0.6 mol) was cooled to about 5
°C, to which were then added alternately 180 ml of 4N NaOH (0.72
mol ) and 69 . 9 ml of DL-2-bromopropionyl bromide ( 0 . 66 mol ) under
cooling and agitation in an ice bath over about 30 minutes.
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The reaction mixture was continuously kept at mild alkalinity.
After the termination of the reaction, the product in white
was filtered and isolated.
The product was dissolved in water, followed by dropwise addition
of 5N HCl to about pH 3. Thereafter, water was removed by
evaporation. While the remaining aqueous solution was
gradually acidified with 5N HCl under cooling, an additional
productin white wasrecovered. These white productsrecovered
were extracted in diethyl ether with a Soxhlet extractor, for
purification.
Yield 30 to 40 0; ~H NMR( O , CDC13) 1.54(d, 3H, NHCHCHj) , 1.91(d,
3H , BrCHCH3 ) , 4 . 4 5 ( q , 1H , NHCHCH3 ) , 4 . 59 ( q , 1H , BrCHCH j ) 6
. 88 ( brs ,
1H, NH).
Continuously, the purified 2-bromopropionyl alanine (19.7 g;
0 . 0881 mol ) and the equimolar NaHC03 ( 7 . 40 g; 0 . 0881 mol ) were
added to 150 ml of dimethylformamide ( DMF ) . Then , the resulting
mixture was refluxed at 60 °C for 24 hours , for intramolecular
cyclization desalting, to recover a cyclic depsipeptide DMO
in white powder (Fig. 3).
DMO was purified via recrystallization twice in chloroform.
Yield 40 to 60 ~;mp 158159°C; 'H NMR( c> , CDC13) 1.54(d, 3H,
NHCHCH3) , 1.62(d, 3H, OCHCH~) , 4.24(q, 1H, NHCH) , 4.91(q, 1H,
OCH), 7.07ppm(brs, 1H, NH).
The synthesis of the terpolymer is now described.
Among the copolymerizable monomers, the cyclic depsipeptide
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(L-DMO) was synthetically prepared from a-amino acid
(L-alanine) and a a-hydroxy acid derivative
(DL-2-bromopropionyl bromide) , and was then purified for use.
Further , lactone ( CL ) was purified by dissolving CL in toluene
and subsequently drying CL with CaHz for 48 hours, and then
subjecting the resulting CL to distillation under reduced
pressure (twice). L-Lactide (L-LA) was purified by
recrystallization in THF and sublimation (twice).
All the polymerization procedures were done in argon atmosphere .
The synthetic scheme of the L-DMO/CL/L-LA terpolymer is shown
in Fig. 4.
The copolymer was prepared as follows.
Given amounts of both the monomers L-DMO and L-LA dissolved
in THF and a toluene solution of a catalytic amount of tin ( I I )
octylate [Sn(Oct)2; 0.2 mol /monomer] are charged in a Schrenk
tube (polymerization container) , from which the solvents THF
and toluene are subsequently trapped and removed under reduced
pressure.
Then, a given amount of the CL monomer is placed in the same
polymerization container, which is then sealed. The sealed
container was immersed in an oil bath at 120 °C, to initiate
the polymerization.
After a given period of time (12 hours), the polymerization
container was taken out of the oil bath and then cooled. The
resulting crude polymerwas dissolved in chloroform and purified
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via reprecipitation (twice) in methanol. Fig. 10 shows the
synthetic conditions of each of the copolymers and the
homopolymers and the yields and molecular weights of the
resulting polymers.
Further, the 1H-NMR data (b, CDC13) of the terpolymer
L-DMO/CL/L-LA (= 8:13:79) is as follows.
1.38 (m, 2H, CH?CH2CH2CHZCH2) , 1.50 (m, 6H, CH3X2(L-DMO) , 1.57(d,
6H, CH, =-< 2(L-LA) ) , 1.68 (m, 4H, CHZCHZCH2CH2CH2) , 2.25-2.45
(splitting in two peaks, 2H, CCH2), 4.60(m, 1H, OCH(L-DMO)),
5.17(q, 3H, OCHX2(L-LA), NHCH(L-DMO)), 6.60ppm(br.m, 1H, NH).
Various physico-chemical properties of the polymers are now
described.
The composition of the copolymer was determined on the basis
of the peak integration ratio of 1H NMR spectrum measured with
a 400 MHz magnetic resonance system ( JEOL JMN-LA400 ) . Based
on the spectrum, the chain sequence ( randomness ) of the copolymer
was deduced as well.
The number average molecular weight (Mn) of the polymer and
the molecular distribution (MW/Mn ) thereof were determined on
the basis of a standard curve prepared from standard polystyrene ,
using GPC 8010 system manufactured by TOSO, Co. , Ltd. [column:
TSK Gel ( G2000HHR + G3000HHR + G4000HHR + G5000HH~ ) , column
temperature of 40 °C and differential refractive index (RI)
meter]. Chloroform was used as the eluent at the flow of 1
mLmin-1.
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The thermal properties of the polymer, namely the glass
transition temperature (Tg), melting point (Tm) and melting
heat (OHm) thereof were measured, using a differential scanning
calorimeter SSC5100 DSC22C manufactured by Seiko Electric Co . ,
Ltd. The measurement was done in nitrogen atmosphere at a
temperature elevation rate of 10 °C/min. Then, the randomness
of the copolymer was also deduced on the basis of the values
of these thermal properties.
The mechanical properties of the polymer ( tensile strength and
elongation at break) were measured, using a tensile tester
RTC-1210 A manufactured by Orientec Co. , Ltd. at a crosshead
speed of 50 mm/min. The measurement was done at least at three
times, to use the average. Additionally, the dumbbell test
piece (parallel length x width x thickness = 12 x 2.65 x 1.46
mm) of a polymer sample was prepared by pressing the polymer
material under heating at 180 to 200 °C for about 5 minutes.
The enzymatic degradation test of the polymer is now described.
The enzymatic degradation test was done in the same manner as
in the related art. The test is now summarized below.
Polymer film (film thickness of about 100 Vim; several tens
milligrams) sealed in polyethylene sheet mesh (mesh size of
about 1 x 1 mm) was incubated ( 37 °C) in a sample tube bottle
placing an enzyme and a buffer ( 50 ml) therein, for degradation.
The enzyme concentration was 1 International Unit ( IU ) per 1
mg of the polymer sample.
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Herein , the buffer containing the enzyme ( degradation solution )
was exchanged to a fresh one every about 40 hours , taking account
of the reduction of the enzyme activity and the contamination
and growth of microorganisms in air.
The degradation level was evaluated on the basis of the changes
of the weight and physico-chemical properties (molecular
weight, composition, and thermal properties) of the polymer
before and after degradation. Proteinase K (derived from
Trl tirachium alburrr; manufactured by Wako Pure Chemical
Industries, Ltd.; activity of 20 IU/mg) was used as one of
proteinases, while Tricine (pH 8.0) was used as Good's buffer.
The polymerization results of the terpolymer L-DMO/CL/L-LA and
each of the homopolymers from the aforementioned tests are shown
in Fig.lO.
In case of the CL homopolymer and the L-LA homopolymer [ poly ( CL )
and poly(L-LA) , respectively] , the polymers of high molecular
weights were recovered at higher yields. Only an L-DMO
homopolymer [poly(L-DMO)] of a low molecular weight was
recovered at a low yield. This may be due to the results of
the ready occurrence of the ester exchange reaction of the
resulting depsipeptide polymer withthe monomer(herein,L-DMO)
and the oligomers and of the back biting reaction thereof causing
molecular chain break.
In case of the terpolymer, alternatively, the reactivity of
L-LA is high under the copolymerization conditions on the basis
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of the copolymer composition ratio. L-DMO is incorporated more
in the copolymer than CL, further, the reactivity of L-DMO is
not so low as CL.
Therefore, the reason why the molecular weight and yield of
the L-DMO homopolymer are low may be ascribed to the ready
occurrence of the ester exchange. The molecular weight and
yield of the copolymer are relatively great, but the values
thereof are gradually decreased as the content of L-DMO is
increased. This also supports the above consideration.
The thermal properties of the resulting terpolymer and each
of the homopolymers are shown in Fig. 11.
As traditionally reported, poly(CL) has softness such that T9
and Tm thereof are about -60 °C and 60 °C, respectively,
nevertheless , a crystallizable polymer with a low melting point .
On the other hand, poly(L-LA) has T9 and Tm at about 60 °C and
180 °C, respectively. The poly(L-LA) is so rigid and fragile
crystallizable polymer with a high melting point, while
poly(L-DMO) is an amorphous glass-like polymer.
In case of the terpolymer, single one Tg and single one Tm are
only observed and their values change as the composition changes ,
suggesting intense randomness. From the respect of balance
between the tensile strength and elongation ( softness ) of the
mechanical properties , appropriately, the Tg value may be around
35 °C.
Fig. 5 depicts the 1H NMR spectrum of the terpolymer L-
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DMO/CL/L-LA ( 8 : 13 : 79 ) . The chart establishes the verification
that the terpolymer is random. In other words , the proton peaks
( f , i ) of the a- and F-methylene in the CL unit are sensitive
to the adjacent comonomer units . It is indicated that because
these peaks are individually split into two (the peak on the
side of high magnetic field corresponds to the homosequence
of CL-CL ; the peak on the side of low magnetic field correspond
to a peak based on the hetero-sequence of L-LA-CL and L-DMO-CL ) ,
the terpolymer is a random copolymer.
Additionally, the reason why the unit L-DMO is introduced
appropriately in the resulting copolymer via copolymerization
at 120 °C lower than the ~'m thereof ( about 170 °C ) is that
the
polymerization of the highly reactive L-LA (with Tm of about
95 °C ) first occurs and the active propagating terminus induces
the ring-opening of the L-DMO (and/or CL), which is then
incorporated randomly in the copolymer.
Fig.l2 depicts the mechanical properties(tensile profile) and
thermal properties of the terpolymer.
The terpolymer in the figure was synthetically prepared freshly
at a large scale, so as to measure these physico-chemical
properties. (So as to modify the softness of the polymer,
mainly, the terpolymer has been synthetically prepared, while
changing the CL amount . ) From the respect of molecular weight
(Mn), all the resulting copolymers had molecular weights of
100 , 000 or more ( 102 , 000 to 158 , 000 ) . It is therefore not so
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much required to consider the influence of the molecular weight
on the physico-chemical properties.
As to the tensile profile, first , the tensile strength is reduced
as the CL content increases or the L-LA amount decreases.
Alternatively, the elongation rapidly increases at the CL amount
of 20 mol o or more, which indicates that the softness of the
copolymer is improved.
The tensile strengths of these terpolymers are larger than that
of a common plastic polyethylene ( PE ) and are of values equal
to or larger than the value of polypropylene ( PP ) . The tensile
strengths thereof are larger than those of biodegradable
plasticsBionolle[polybutylenesuccinate(PBSU);manufactured
by Showa Polymer Co., Ltd.] and Biopol [P(3HB-co-3HV);
manufactured by Nippon Monsanto Co., Ltd.].
The break elongation of a sample at a CL content of 20 mol o
or more is far larger than that of Biopol and is at the same
level as or a higher level than those of PE , PP and Bionolle .
Like the case in Fig.ll, alternatively, both the thermal
properties Tm and nHm of the terpolymers are decreased when
the CL amount is increased. It is shown that all the terpolymers
are crystallizable polymers with Tm of 100 °C or more.
Judging fromthese mechanical propertiesand thermal properties,
the terpolymer L-DMO/CL/L-LA ( 4 : 20 : 76 ) ( Tg = 34 . 3 °C ) at a CL
content of 20mo1 ~ or more has a good balance in the
physico-chemical properties.
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Before examination of the enzymatic degradation level, then,
a hydrolysis test with a degradation solution containing a buf fer
alone was done (Fig. 6). Herein, the L-DMO homopolymer was
not used at the hydrolysis test because the homopolymer was
water-soluble. As shown in the figure, the hydrolysis level
is decreased as the CL unit amount is increased but is
alternatively increased as the L-DMO unit is increased. Thus,
the increasing order of hydrophobicity is L-DMO < L-LA < CL
unit. However, the weight loss of a terpolymer at the maximum
is about 10 ~ 200 hours later.
Fig. 7 shows the enzymatic degradation levels of the terpolymer
and each of the homopolymers with proteinase K.
In case of the homopolymers , poly ( L-LA ) is decomposed at some
extent. Poly(CL) more readily decomposable from the respect
of thermal properties hardly lost the weight within 200 hours .
This may be ascribed to the high substrate specificity of the
enzyme to polymers with shorter alkyl chain lengths between
ester bonds and with side chains [ herein , poly( L-LA ) with lactoyl
group t -O-CH ( CH3 ) -CO- } ] but no specificity thereof to poly ( CL )
with the linear ethylene chain between ester bonds of a
relatively long length. In case of the terpolymer,
alternatively,the degradationlevelismoreincreased compared
with the poly(L-LA), when the content of the L-DMO unit is
increased. The substrate specificity of the enzyme to the L-DMO
unit with lactoyl group alike may be a big factor for this
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increase.
It is suggested as an additional cause that the molecular weight
(Mn ) and thermal properties ( Tm, OHm ) of the terpolymer are reduced ,
compared with poly(L-LA) (Fig.ll).
In any way, the terpolymer L-DMO/CL/L-LA could get an improved
enzyme degradation level with proteinase K while the terpolymer
L-DMO/CL/L-LA relatively retained the thermal and mechanical
properties of poly(L-LA).
So as to speculate the degradation mechanism with the enzyme ,
continuously, the changes of various physico-chemical
properties of the terpolymer L-DMO/CL/L-LA ( 8 : 8 : 84 ) before and
after degradation with proteinase K were examined (Fig. 13 ) .
As shown in Fig. 13, the result is that the content of the L-DMO
unit in the residual polymer was highly reduced as the
degradation proceeded. As described above, the enzyme has
substrate specificity to both the units L-LA and L-DMO. Because
the polymer domain containing the L-LA unit is crystallizable,
the degradation level of the amorphous hydrophilic region highly
containing the L-DMO unit is elevated, possibly leading to the
decrease of the composition ratio.
It is also suggested that because the amount of the CL unit
hardly decomposable was also decreased, the CL unit existed
adjacent to the L-DMO unit and the ester bond between the two
units was broken with the enzyme. The molecular weight (M~)
of the polymer decreased as the degradation proceeded, while
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the distribution (MW/M~) was likely to be enlarged. Thus, it
is indicated that the degradation with the enzyme randomly
progressed in the inside of the polymer film,
Because the thermal properties (2'm,OHm) of the polymer were
elevated as the progress of the degradation, further, it is
indicated that the degradation of the amorphous hydrophilic
region highly containing the L-DMO unit preferentially
occurred.
Based on the results with NMR and the measurement of the thermal
properties, it was shown that the resulting copolymer was a
random copolymer.
This indicates that the degradation rate remarkably increased
via the addition of depsipeptide, without any loss of the
mechanical strength and softness.
Further, Fig. 7 shows the enzymatic degradation properties of
the copolymer with the depsipeptide unit.
Additionally, the above description has been done, provided
that the lactide is L-lactide. L-Lactide and the enantiomer
D-lactide are polymerized together in combination, to form a
stereo complex with improved thermal properties such as melting
point.
Still further, the change of the glass transition temperature
can impart free formation potency.
Therefore, a bicopolymer produced by copolymerizing together
a depsipeptide and L-lactide to ring-open and polymerize the
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depsipeptide may be satisfactory, other than the terpolymer.
Additionally, a lactide with a formed stereo complex as produced
by copolymerizing a combination of L-lactide and the enantiomer
D-lactide together is copolymerized with a depsipeptide, to
form a stereo complex of a copolymer of the ring-opened and
polymerized depsipeptide.
Second embodiment
Tn case of the copolymer of a depsipeptide with
r-caprolactone, the structure of the copolymer with the peptide
unit is shown in Fig. 2. U expresses the depsipeptide unit.
The procedure also imparted mechanical strength and increased
degradation rate as in the first embodiment.
So as to elucidate the influence of the depsipeptide unit in
the copolymer with the peptide units, further, the R group in
the side chain in the depsipeptide was modified into methyl
group, isopropyl group or isobutyl group, to examine the
influence.
Fig. 8 shows the degradation levels of the copolymers with the
depsipeptide units.
The figure shows that the degradation level is in the order
of methyl group » isopropyl group > isobutyl group. It is
shown that the increase of the bulkiness of the side chain
decreases the degradation level.
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Third embodiment
3-Isopropyl-6-methyl-2,5-morpholine-dione (PMO) used as a
depsipeptide was copolymerized with-caprolactone,to prepare
a copolymer, where the depsipeptide was ring-opened and
polymerized.
Then, the changes of the thermal properties and degradation
rate in case of the change of the depsipeptide amount were
examined.
Fig. 14 shows the relation of the thermal properties , while Fig. 9
shows the relation of the degradation rate.
According to the results, the glass transition temperature ( T,~)
was elevated as the depsipeptide amount increased. At the
amount of ~-caprolactone at 20mo1 ~ or less, the melting point
( Tn,) and the heat of fusion(OHm) were observed, indicating that
the resulting copolymer was crystallizable.
The degradation rate was elevated as the amount of the
depsipeptide increased.
Herein, the description in the individual embodiments has been
done, exemplifying poly E-caprolactone and polylactic acid as
the bio-absorbable polymers. However, the bio-absorbable
polymers are not limited to them. Any bio-absorbable polymer
may be satisfactory, including for example polydioxanone,
trimethylene carbonate and copolymers of two or more thereof .
Industrial Applicability
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In accordance with the invention described in detail above,
a copolymer with a depsipeptide unit, as produced by
copolymerizing a cyclic depsipeptide with a bio-absorbable
monomer, can advantageously be modified into a biodegradable
bio-absorbable material with adjusted dynamic properties and
degradation properties for clinical practice.
Further, advantageously, the modification of the peptide
unit with alkyl groups can adjust the dynamic properties and
the degradation properties.
