JP4781332B2 - Polyphosphate compound having temperature responsiveness and method for producing the same - Google Patents

Description

  The present invention relates to a polyphosphate compound that gels in response to temperature and has biodegradability and a method for producing the same.

  In general, degradable plastics that decompose and disappear by hydrolysis and enzymatic decomposition are used as materials for environmental protection and medical devices. However, aliphatic polyesters such as polylactic acid and polyglycolic acid, which are general degradable polymers, have a very long degradation time due to their high crystallinity and hydrophobicity. Especially when used as medical devices, they remain in vivo. Is a problem. Therefore, a new degradable polymer that is excellent in degradability and does not harm the living body has been desired.

  In order to solve this problem, a polyphosphate having a polymerizable group obtained by anionic ring-opening polymerization of a cyclic phosphate compound having various alkyl chains and a cyclic phosphate compound having a polymerizable group has excellent degradability. It has been reported to have (Patent Document 1).

  Biodegradable polymers using aliphatic polyesters such as polylactic acid have also been proposed (for example, Non-Patent Documents 1 to 3), but their molecular structure is difficult to control and is not as widespread as PNIPAAm.

By the way, poly (N-isopropylacrylamide) (PNIPAAm) is the most common temperature-sensitive polymer, and is known to exhibit a lower critical solution temperature (LCST) around 32 ° C. (Non-Patent Documents 4 to 4). 5). However, since PNIPAAm has an acrylamide skeleton, it does not exhibit biodegradability, and its use in fields related to living bodies is limited. In addition, the safety of the polymer itself is also considered problematic.
JP 2004-244603 A Agrawal CM, Niederauer GG, Micallef DM, Athanasiou KA.The use of PLA-PGA polymers in orthopedics.In: Encyclopedic handbook of biomaterials and bioengineering, Part A. New York: Marcel Dekker; 1995.p 1055-1089. Miller RA, Brady JM, Curtright DE. Degradation rates of oral resorbable implants (polylactates and polyglycolates): Rate modification with change PLA / PGA copolymer ratios.J Biomed Mater Res 1977; 11: 711-719. Lewis DH. Controlled release of bioactive agents from lactide / glycolide polymers.In: Chain M, Langer R, editors.Biodegradable polymers as drug delivery systems.New York: Marcel Dekker; 1990. p 1-41. Heskins, M .; Guillent, JE; James, EJ Macromol. Sci. Chem. 1968, A2, 1441-1445. Takei, YG; Aoki, T .; Sanui, K .; Ogata, N .; Okano, T .; Sakurai, Y. Bioconjug. Chem. 1993, 4, 341-346.

  An object of this invention is to provide the novel copolymer compound which has temperature responsiveness, and has biodegradability, and its manufacturing method.

  As a result of intensive studies to solve the above problems, the present inventor has found that a copolymer compound having a biodegradable phosphate ester bond in the main chain and a hydrophilic group different in the side chain has a temperature response. It was found to have sex. Based on this knowledge, this has been further developed to complete the present invention.

  That is, this invention provides the following polyphosphate compound and its manufacturing method.

  Item 1. General formula (3):

Wherein R ¹ is a C1-3 alkyl group, R ² is a C3-5 alkyl group, and R ¹ and R ² are different. N and m are the same or different and are 1 to 3 X and y are the mole percentages of each unit, x / y is 99.9 / 0.1 to 20/80, and the weight average molecular weight is 1,000 to 1,000,000. (It is not limited to the order of arrangement.)
Polyphosphate represented by

Item 2. In Formula (3), an alkyl group of ^{R 1} is C1-2, ^{R 2} is as defined in claim 1 is an alkyl group of C3~4 polyphosphate.

  Item 3. Item 3. The polyphosphate according to Item 1 or 2, wherein in the general formula (3), x / y is 99.9 / 0.1 to 80/20.

  Item 4. General formula (3):

Wherein R ¹ is a C1-3 alkyl group, R ² is a C3-5 alkyl group, and R ¹ and R ² are different. N and m are the same or different and are 1 to 3 X and y are the mole percentages of each unit, x / y is 99.9 / 0.1 to 20/80, and the weight average molecular weight is 1,000 to 1,000,000. (It is not limited to the order of arrangement.)
A process for producing a polyphosphate represented by the general formula (1):

(In the formula, R ¹ and n are the same as above.)
A compound represented by formula (2):

(In the formula, R ² and m are the same as above.)
And a compound represented by formula (1) in the presence of a polymerization catalyst.

  Item 5. A medical material containing the polyphosphate according to any one of Items 1 to 3.

  The polyphosphate compound of the present invention is a biodegradable polymer having temperature responsiveness. Since the polymer having a phosphate ester bond main chain is enzymatically and non-enzymatically degraded, it can be used in vivo, which could not be realized with PNIPAAm. That is, application to a single drug or a material for tissue engineering is possible. In addition, the temperature responsiveness can be controlled by changing the combination of the hydrophobic groups in the side chains of the polyphosphate compound. From the above, the polyphosphate compound of the present invention is suitably used as a medical material.

  Furthermore, since the polyphosphate compound of the present invention has a simple production method, the molecular structure can be easily controlled, and the chemical structure of the side chain can be modified, so that polymers having various properties can be designed. Become.

1. Polyphosphate Compound The polyphosphate compound of the present invention has the general formula (3):

Wherein R ¹ is a C1-3 alkyl group, R ² is a C3-5 alkyl group, and R ¹ and R ² are different. N and m are the same or different and are 1 to 3 X and y are the mole percentages of each unit, x / y is 99.9 / 0.1 to 20/80, and the weight average molecular weight is 1,000 to 1,000,000. (It is not limited to the order of arrangement.)
It is a compound represented by these.

Examples of the C1-3 alkyl group represented by R ¹ include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. Among these, a methyl group and an ethyl group are preferable.

Examples of the C3-5 alkyl group represented by R ² include n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, and isopentyl group. . Among these, n-propyl group and isopropyl group are preferable.

It is important that R ¹ and R ² are different, and R ² is preferably more hydrophobic than R ^1, that is, preferably has a larger number of carbon atoms.

  n and m are the same or different and are an integer of 1 to 3, preferably an integer of 1 or 2, and more preferably n and m are both 1.

  x and y are mole percentages of each unit, x is 99.9-20, preferably 99.9-30, more preferably 99.9-50, y is 0.1-80, preferably 0.1-70, more preferably 0.1- 50. When expressed in x / y, it is 99.9 / 0.1 to 20/80, preferably 99.9 / 0.1 to 30/70, more preferably 99.1 / 0.1 to 50/50. Within such a range, suitable temperature responsiveness and biodegradability are imparted to the polymer of the present invention.

  In particular, a polymer that has an LCST at a temperature lower than the body temperature and forms a gel state in the vicinity of the body temperature (about 37 ° C.) when used for use in a living body is preferable. y is 98/2 to 20/80, preferably 95/5 to 30/70, more preferably 90/10 to 50/50.

X and y can be determined from ¹ H-NMR. As described above, the arrangement of each unit of x and y may be random or block.

  The weight average molecular weight (Mw) of the polyphosphate compound is 1,000 to 1,000,000, preferably 1,000 to 500,000, more preferably 5,000 to 100,000. The number average molecular weight (Mn) is 1,000 to 1,000,000, preferably 1,000 to 500,000, more preferably 5,000 to 100,000, and the ratio of the weight average molecular weight to the number average molecular weight (Mw / Mn), which is an index of molecular weight distribution, is 1.0 to 3.0. , Preferably, it is 1.0-2.0, More preferably, it is 1.0-1.5. The number average molecular weight and the weight average molecular weight can be measured using a known method such as GPC.

In particular, R ¹ is an ethyl group, R ² is an isopropyl group, n and m are both 1, x / y is 99.9 / 0.1 to 30/70 (particularly 90/10 to 50/50), In the case of a polymer having a weight average molecular weight of 1,000 to 500,000, since the LCST is about 20 to 40 ° C. (particularly about 20 to 35 ° C.), it is suitable as an injectable polymer to be embedded in a living body. For example, the polyphosphate compounds of the present invention are stored below the gel temperature and are administered parenterally by intramuscular, intraperitoneal, subcutaneous, or similar injection methods and gelled near body temperature and remain in the body. Can be made.
2. Production Method of Polyphosphate Compound The polyphosphate compound of the present invention can be produced, for example, as follows.

(Wherein R ¹ , R ² , n, m, x and y are the same as above).
The compounds represented by the above (1) and (2) are subjected to ring-opening polymerization in the presence of a polymerization catalyst to produce the compound represented by the above (3).

  The compounds represented by the above (1) and (2) can be produced according to the description in Patent Document 1, for example.

  The molar ratio of the compounds represented by the above (1) and (2) is prepared so as to be the molar percentage of the above x and y. Specifically, for example, x / y is 99.9 / 0.1 to 20/80, preferably 99.9 / 0.1 to 30/70.

  Examples of the polymerization catalyst include alkyl metals such as triisobutylaluminum, butyllithium, trimethylaluminum, triisobutoxyaluminum, and tin octoate. Triisobutylaluminum is preferable. The usage-amount of a polymerization catalyst is 0.01-10 mol% with respect to the total mol of the compound represented by said (1) and (2), Preferably it is 0.05-1 mol%.

  The polymerization reaction can be performed in the presence of a solvent or without a solvent. When using a solvent, for example, tetrahydrofuran, dioxane, chloroform, acetonitrile, diethyl ether and the like can be used. These solvents may be used alone or in combination of two or more. This polymerization reaction is preferably carried out without a solvent.

  The polymerization reaction is usually carried out in a sealed reaction vessel. The polymerization reaction conditions are not particularly limited, but usually the reaction temperature is -20 to 100 ° C, preferably -20 to 0 ° C, the reaction pressure is 100 mmHg to 760 mmHg, preferably 500 mmHg to 760 mmHg, and the reaction time is 2 ~ 48 hours.

The polyphosphate compound represented by the formula (3) thus obtained is used as it is or after the reaction solvent is partially or completely distilled off, the solvent (for example, ethanol) in which the polyphosphate compound can be dissolved. It can be purified as a precipitate by adding it to a solvent (eg, diethyl ether, hexane, etc.) in which the polyphosphate compound does not dissolve. Alternatively, it can also be isolated and purified by column chromatography, extraction, dialysis, ultrafiltration, adsorption treatment, or the like.
3. Properties and Uses of Polyphosphate Compounds The polyphosphate compounds of the present invention are biodegradable and temperature responsive. Here, the temperature responsiveness generally refers to a property in which an aqueous solution of a compound exhibits a reversible sol-gel transition phase separation behavior with a lower critical solution temperature (LCST) as a boundary. Specifically, when heated to a temperature of LCST or higher, it becomes cloudy and becomes a gel state, and when cooled to a temperature lower than that, it dissolves again and returns to a transparent sol state.

  As used herein, the LCST of a polyphosphate compound is defined as the temperature at the midpoint of the solution permeability transition state using JASCO software.

  In the polyphosphate compound of the present invention, LCST usually exists in a range of about 20 to 40 ° C., and LCST can be easily adjusted in such a range. In particular, the above properties are suitably exhibited in the case of the compound represented by the general formula (3). For example, the LCST of polyphosphate compounds tends to decrease as the mole percent of the unit represented by y, which is more hydrophobic than the unit represented by x, increases. This is considered to be because the dehydration of the polymer is likely to occur even at a low temperature as the number of hydrophobic units increases.

  The polyphosphate compound of the present invention can be used in a wide range of applications. For example, it can be used as a medical material together with a drug. Further, it can be applied as a scaffold for regenerative medicine, a cell culture substrate and the like.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

For the analysis and identification of the compounds, 1H-NMR, 31P-NMR, and infrared spectroscopic spectrum (FT-IR) gel permeation chromatography (GPC) were used. GPC used polystyrene as a standard substance.
[Example 1] Synthesis of graft copolymer

  2-Ethoxy-2-oxo-1,3,2-dioxaphosphorane (EP) and 2-isopropyl-2-oxo-1,3,2-dioxaphosphorane (IPP) were treated with Macromol. Biosci. 2003. , 3, 238-242.

A copolymer consisting of EP and IPP (represented as “PI _y E _x P”, EP x mol% and IPP y mol%) is subjected to a ring-opening polymerization reaction using triisobutylaluminum (iBu ₃ Al) as a catalyst. It was. In the polymerization reaction, EP and IPP are mixed at a predetermined ratio so that the total amount of monomers is 0.025 mol, and 15 μl of iBu ₃ Al is used, and 2 at room temperature (25 ° C.) and normal pressure (760 mmHg) without solvent. A copolymer (PI _y E _x P) was produced by a time-homogeneous reaction.

The obtained polymer was dissolved in ethanol and purified by precipitation by adding diethyl ether thereto. The range of the weight average molecular weight (Mw) measured by gel permeation chromatography (GPC) was 1.2 × 10 ^{4 to} 1.5 × 10 ⁵ g / mol. Moreover, molecular weight distribution (Mw / Mn) was less than 1.3. The molar percentages of IPP (y) and EP (x) in the copolymer (PI _y E _x P) were calculated from ¹ H-NMR spectrum and were 24 mol% and 76 mol%, respectively.

¹ H NMR (270 MHz, CDCl ₃ ): δ = PI _y E _x P: 1.21-1.47 (m; -CH ₃ , 6H in IPP and 3H in EP), 3.95-4.20 (m; -CH _2- , 2H in EP and -OCH ₂ CH ₂ O-, 4H in backbone), 4.58 (m; -CH (CH ₃ ) ₂ in IPP, 1H).
The polymerization ratio (r ₁ / r ₂ ) of IPP and EP was determined to be 0.48 / 2.23 by the Fineman-Ross method. EP was much more reactive than IPP.
r ₁ / r ₂ is the copolymerization reactivity ratio, and represents the change in y / x of the copolymer relative to the y / x of the feed. In other words, the larger the value, the higher the reactivity, and the EP composition is higher in the polymer than in the charged.
y / x is a variable and corresponds to Figure 2.
[Example 2]
LCST was measured using a 0.5 wt% / L aqueous solution of a copolymer (PI ₂₄ E ₇₆ P) containing ₂₄ mol% IPP and 76 mol% EP produced in Example 1 (see FIG. 1). The polymer solution was irradiated with visible light of 500 nm using an ultraviolet-visible absorptiometer, and the change in transmittance was measured.

The solution was clear at 15 ° C. and became opaque at 40 ° C. (turbidity occurred). The LCST of this copolymer was 31 ° C. from the midpoint temperature of the transmission transition state using JASCO software. This revealed that the copolymer exhibited phase separation behavior under physiological conditions.
[Example 3]
The effect of the monomer unit content on the LCST of the copolymer was investigated. The LCST of poly (EP) was 39 ° C., and was confirmed to decrease linearly as the IPP content increased (see FIG. 2). IPP is more hydrophobic than EP, and IPP is insoluble in water at about 5 ° C. Therefore, addition of hydrophobic IPP units resulted in dehydration of the polymer. That is, it was found that the LCST of the obtained copolymer can be easily controlled by changing the composition ratio.

The temperature responsiveness of this copolymer was observed at physiological conditions. Temperature responsiveness under physiological conditions is effective for drug delivery and tissue engineering applications.
[Example 4]
Regarding the copolymer (PI ₂₄ E ₇₆ P) produced in Example 1, a change in light transmittance of the aqueous solution was observed in accordance with a change in temperature repeatedly (see FIG. 3). The polymer solution was irradiated with visible light of 500 nm using an ultraviolet-visible absorptiometer, and the change in transmittance was measured.

  Hysteresis of transmittance change due to temperature change was observed, and a curve corresponding to temperature was observed regardless of the number of repetitions. This shows that the copolymer is completely dissolved at a low temperature.

In addition, the hydrodynamic radius (R _h ) of the copolymer (PI ₂₄ E ₇₆ P) in the aqueous solution was measured using the Malvern dynamic light scattering technique, which was less than 20 ° C. , R _h was 6.7 ± 0.1 nm, and at 50 ° C. the polymer was associated and R _h exceeded 6 μm. It was found that the phase separation behavior of the polymer was easily reproduced.

It is a photograph at 15 ℃ and 40 ° C. of an aqueous solution of the copolymer of Example _{2 (PI 24 E 76 P)} . It is a graph showing the effect of the content of IPP in LCST of the copolymer of Example _{3 (PI y E x P)} . It is a graph showing the change in light transmittance in the aqueous solution at a temperature change of the copolymer (PI 24 _{E 76 P)} in Example 4.

Claims (5)

General formula (3):

Wherein R ¹ is a C1-3 alkyl group, R ² is a C3-5 alkyl group, and R ¹ and R ² are different. N and m are the same or different and are 1 to 3 X and y are the mole percentages of each unit, x / y is 99.9 / 0.1 to 20/80, and the weight average molecular weight is 1,000 to 1,000,000. (It is not limited to the order of arrangement.)
Polyphosphate represented by In Formula (3), an alkyl group of ^{R 1} is C1-2, as defined in claim 1 ^{R 2} is an alkyl group of C3~4 polyphosphate. The polyphosphate according to claim 1 or 2, wherein in the general formula (3), x / y is 99.9 / 0.1 to 50/50. General formula (3):

Wherein R ¹ is a C1-3 alkyl group, R ² is a C3-5 alkyl group, and R ¹ and R ² are different. N and m are the same or different and are 1 to 3 X and y are the mole percentages of each unit, x / y is 99.9 / 0.1 to 20/80, and the weight average molecular weight is 1,000 to 1,000,000. (It is not limited to the order of arrangement.)
A process for producing a polyphosphate represented by the general formula (1):

(In the formula, R ¹ and n are the same as above.)
A compound represented by formula (2):

(In the formula, R ² and m are the same as above.)
And a compound represented by formula (1) in the presence of a polymerization catalyst. The medical material containing the polyphosphate in any one of the said Claims 1-3.

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