JP2006111585A - Sustained release composition and sustained releasing method therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a sustained release composition which can control the releasing process of an active substance so as to conform to the purpose. <P>SOLUTION: The sustained release composition containing at least a hydrogel-forming polymer which can form a hydrogel having a sol-gel transition temperature; a dispersing liquid; and the active substance, and also containing at least fine particles capable of revealing the sustained releasing property of the active substance by themselves, is provided. This composition is in a flowable sol state at a temperature lower than the sol-gel transition temperature, and reversibly in a hydrogel state at a temperature higher than the sol-gel transition temperature. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a sustained release composition containing a hydrogel. More specifically, the present invention can be easily applied to a target site in a low-temperature fluid state, becomes a gel at the use temperature (for example, body temperature), and gradually releases the active substance inside the gel over a certain period of time. Relates to a sustained release composition.

  The sustained-release composition of the present invention can be applied without particular limitation to uses where such sustained release is beneficial. For convenience of explanation, a physiologically active substance such as a drug is used here as an active substance. Background art related to aspects of the present invention will be mainly described.

  In recent years, the drug delivery system (Drug Delivery System) is a drug administration method that provides the required amount of drug to the affected area for the required time and maximizes its efficacy and minimizes side effects. DDS) is being actively developed. The present inventors have already proposed a drug carrier containing a polymer compound that gels at a temperature lower than the body temperature and shows water solubility at a temperature lower than the gelation temperature (Patent Document 1: JP-A-5-255119). DDS (drug-diffusion process of drug molecules with simple drug sustained release) that combines this drug carrier with drugs such as anticancer drugs, various hormone drugs, immunostimulants such as interferon and interleukin, narcotic antagonists, and anesthetics Since it is liquid at the administration temperature, it is easy to inject by oral, injection, catheter, etc., and local administration and sustained release of the drug to instantly gel in the body's tube or in vivo. Was possible.

  However, in recent years, there has been a demand for a sustained-release composition that can be sustained-released for a longer time or has a stronger drug release control from the viewpoint of medicinal effect or its sustained effect.

JP-A-5-255119

  An object of the present invention is to provide a sustained-release composition that solves the above-mentioned problems.

  Another object of the present invention is to provide a sustained release composition capable of sustained release of an active substance for a longer period of time.

  Another object of the present invention is to provide a sustained release composition capable of controlling the release process of an active substance so as to meet the purpose.

  As a result of various studies, the present inventors have combined the hydrogel with the fine particles containing at least the active substance, so that the active substance can be slowly released independently of the process of free diffusion in the hydrogel. And found that it is extremely effective in achieving the above objective.

The sustained-release composition of the present invention is based on the above findings, and more specifically, a hydrogel-forming polymer capable of forming a hydrogel having a sol-gel transition temperature; a dispersion liquid; and a fine particle containing an active substance A fluid sol state at a temperature lower than the sol-gel transition temperature, and a hydrogel state reversibly at a temperature higher than the sol-gel transition temperature.
According to the present invention, the polymer further comprises a hydrogel-forming polymer capable of forming a hydrogel having a sol-gel transition temperature; a dispersion liquid; and fine particles containing an active substance; and a temperature lower than the sol-gel transition temperature. Then, a sustained release composition that becomes a fluid sol state and reversibly becomes a hydrogel state at a temperature higher than the sol-gel transition temperature,
Placing the sustained release composition at a temperature lower than the fluid sol-gel transition temperature at an application site where sustained release should be developed;
There is provided a sustained release method characterized in that the active substance is sustainedly released from the sustained release composition by gelation at a temperature higher than the sol-gel transition temperature.

  If the sustained-release composition of the present invention is used, it can be easily injected or placed in a target site or location (for example, the inside of a living body or the surface of a living body) in a sol state at a temperature lower than the sol-gel transition temperature. Since it can be made into a hydrogel state having no fluidity by gelling at the use temperature (for example, body temperature), it can be left at the target site for a long time.

  In the present invention, the diffusion rate of fine particles containing an active substance in a hydrogel can be extremely slow. For example, at the use temperature of a sustained-release composition (for example, human body temperature (about 37 ° C.)) It is also possible not to substantially diffuse.

  According to the knowledge of the present inventor, the release of the active substance from the sustained release composition of the present invention can be controlled by a combination of the following four types of release processes.

1) Diffusion process of active substance in hydrogel,
2) Diffusion process of fine particles containing active substance in hydrogel,
3) Active substance release process from fine particles containing active substance,
4) Hydrogel disintegration process.

  As described above, the sustained-release composition of the present invention can be controlled by a combination of the above-described four types of release processes. Therefore, an ideal release behavior of an active substance suitable for the purpose of using the composition ( For example, it is possible to design a near ideal release behavior of the active substance that was not possible with conventional sustained release compositions.

  Hereinafter, the present invention will be described more specifically with reference to the drawings as necessary. In the following description, “parts” and “%” representing the quantity ratio are based on mass unless otherwise specified.

(Hydrogel)
The hydrogel of the present invention contains at least a hydrogel-forming polymer having a sol-gel transition temperature. The hydrogel exhibits a thermoreversible sol-gel transition that becomes a sol state at a lower temperature and a gel state at a higher temperature.

  The “hydrogel-forming polymer” constituting the hydrogel of the present invention is a hydrogel having a cross-linking structure or a network structure and holding a dispersion liquid such as water based on the structure. A polymer having properties that can be formed. The “hydrogel” refers to a gel containing at least a crosslinked or network structure made of a polymer and water supported or held in the structure (dispersed liquid).

(Dispersed liquid)
The “dispersed liquid” retained in the crosslinked or network structure is not particularly limited as long as it is a liquid containing water as a main component. More specifically, the dispersion liquid may be water itself, and may be either an aqueous solution and / or a water-containing liquid. The water-containing liquid preferably contains 80 parts or more, more preferably 90 parts or more of water with respect to 100 parts of the whole water-containing liquid.

  Unless it is contrary to the objective of this invention, the said dispersion liquid may contain the organic solvent (For example, hydrophilic solvents, such as ethanol which is compatible with water) with a predetermined | prescribed content.

(Sol-gel transition temperature)
In the present invention, the definition and measurement of “sol state”, “gel state” and “sol-gel transition temperature” are described in the literature (H. Yoshioka et al., Journal of Macromolecular Science, A31 (1), 113 (1994)). That is, the dynamic elastic modulus of the sample at an observation frequency of 1 Hz is measured by gradually changing the temperature from the low temperature side to the high temperature side (1 ° C./1 min), and the storage elastic modulus ( The temperature at which G ′, the elasticity term) exceeds the loss modulus (G ″, viscosity term) is defined as the sol-gel transition temperature. In general, the state of G ″> G ′ is defined as sol, and the state of G ″ <G ′ is defined as gel. In measuring the sol-gel transition temperature, the following measurement conditions can be preferably used.

<Dynamic / loss elastic modulus measurement conditions>
Measuring device (trade name): Stress-controlled rheometer AR500, manufactured by TA Instruments Inc. Sample solution (or dispersion) concentration (however, as the concentration of “hydrogel-forming polymer having sol-gel transition temperature”) : 10 (weight)%
Amount of sample solution: about 0.8 g
Shape and dimensions of measurement cell: acrylic parallel disk (diameter: 4.0 cm), gap: 600 μm
Measurement frequency: 1Hz
Applied stress: in the linear region.

  In an embodiment in which the composition of the present invention is applied to a human living body, the sol-gel transition temperature is preferably higher than 0 ° C. and lower than 37 ° C. from the viewpoint of preventing thermal damage to living tissue. It is preferably higher than 5 ° C and not higher than 35 ° C (particularly not lower than 10 ° C and not higher than 33 ° C).

  Such a hydrogel-forming polymer having a suitable sol-gel transition temperature is easily selected from the specific compounds described below according to the screening method (sol-gel transition temperature measurement method) described above. be able to. In a series of operations in which the sustained-release composition of the present invention is placed at a target site in a living body and the active substance is released slowly, the above-described sol-gel transition temperature (a ° C.) is taken as the temperature of the living body (b ° C.). It is preferable to set the temperature between the temperature (c ° C.) at the time of cooling for injection into the target site of the living body. That is, it is preferable that there is a relationship of b> a> c between the above three temperatures a ° C., b ° C., and c ° C. More specifically, (b-a) is preferably 1 to 36 ° C, more preferably 2 to 30 ° C, and (ac) is 1 to 35 ° C, more preferably 2 to 30 ° C. Is preferred.

(Followability for operation of sustained-release composition)
The hydrogel based on the sustained-release composition of the present invention exhibits a solid behavior at a higher frequency from the point of balance of conformity to the morphological change of the living tissue, and on the other hand, at a lower frequency. On the other hand, it is preferable to exhibit a liquid behavior. More specifically, the followability to the operation of the hydrogel can be suitably measured by the following method.

(Measuring method for tracking performance)
The sustained-release composition of the present invention (1 mL as a hydrogel) containing a hydrogel-forming polymer is put in a test tube having an inner diameter of 1 cm in a sol state (temperature lower than the sol-gel transition temperature), and the sustained-release composition The test tube is held for 12 hours in a water bath at a temperature sufficiently higher than the sol-gel transition temperature of the product (for example, a temperature about 10 ° C. higher than the sol-gel transition temperature) to gel the hydrogel.

Next, the time (T) until the solution / air interface (meniscus) is deformed by its own weight when the test tube is turned upside down is measured. Wherein 1 / T said hydrogel for an operating frequency lower than ^{(sec -1)} behaves as a liquid, for operation of the frequency higher than 1 / T ^{(sec -1),} the hydrogel as a solid Will behave. In the case of the hydrogel of the present invention, T is 1 minute to 24 hours, preferably 5 minutes to 10 hours.

(Steady flow viscosity)
The gel property of the hydrogel based on the sustained-release composition of the present invention can be suitably measured by measuring the steady flow viscosity. The steady flow viscosity η (eta) can be measured, for example, by a creep experiment. In the creep experiment, a constant shear stress is applied to the sample and the temporal change of shear strain is observed. Generally, in the creep behavior of a viscoelastic body, the shear rate initially changes with time, but thereafter the shear rate becomes constant. The ratio between the shear stress and the shear rate at this time is defined as the steady flow viscosity η. This steady flow viscosity is sometimes referred to as Newtonian viscosity. Here, however, the steady flow viscosity must be determined within a linear region that is largely independent of shear stress.

Specifically, a stress-controlled viscoelasticity measuring device (AR500, manufactured by TA Instruments) is used as a measuring device, an acrylic disk (diameter 4 cm) is used as a measuring device, and a sample thickness is 600 μm for at least 5 minutes. Observe the above measurement time creep behavior (delay curve). Sampling time is once per second for the first 100 seconds and then once every 10 seconds. In determining the applied shear stress (stress), the shear stress is applied for 10 seconds and set to the lowest value at which the deviation angle is detected at 2 × 10 ⁻³ rad or more. The analysis employs at least 20 measured values after 5 minutes. The hydrogel based on the sustained-release composition of the present invention preferably has a η of 5 × 10 ^{3 to} 5 × 10 ⁶ Pa · sec at a temperature about 10 ° C. higher than the sol-gel transition temperature. It is preferable that it is 8 * 10 < ³ > -2 * 10 < ⁶ > Pa * sec, especially 1 * 10 < ⁴ > Pa * sec or more and 1 * 10 < ⁶ > Pa * sec or less.

When the η is less than 5 × 10 ³ Pa · sec, the fluidity is relatively high even in short-time observation, and it is easy to move from the target site in the living body. On the other hand, when η exceeds 5 × 10 ⁶ Pa · sec, the gel tends to hardly exhibit fluidity even when observed for a long time, and the followability of the sustained-release composition to the deformation of the living body becomes insufficient. Further, when η exceeds 5 × 10 ⁶ Pa · sec, the possibility that the gel exhibits brittleness increases, and after a slight pure elastic deformation, a tendency to easily cause brittle fracture that breaks at once is increased.

(Dynamic elastic modulus)
The gel-like properties of the hydrogel based on the sustained-release composition of the present invention can be suitably measured also by the dynamic elastic modulus. When a strain γ (t) = γ ₀ cos ωt (t is time) with an amplitude γ ₀ and a frequency ω / 2π is given to the gel, σ (t) where σ ₀ is a constant stress and δ is a phase difference ) = Σ ₀ cos (ωt + δ) is obtained. When | G | = σ ₀ / γ ₀ , the ratio (G ″ / G ′) of the dynamic elastic modulus G ′ (ω) = | G | cos δ and the loss elastic modulus G ″ (ω) = | G | sin δ ) Is an index representing gel-like properties.

The hydrogel based on the sustained release composition of the present invention behaves as a solid with respect to strain of ω / 2π = 1 Hz (corresponding to fast operation) and strain of ω / 2π = 10 ⁻⁴ Hz ( Behaves as a fluid for slow motion). More specifically, the hydrogel based on the sustained release composition of the present invention preferably exhibits the following properties (for details of such elastic modulus measurement, see, for example, literature: Ryohei Oda, edited by Modern Industrial Chemistry 19, page 359, Asakura Shoten, 1985).

  When ω / 2π = 1 Hz (frequency at which the gel behaves as a solid), (G ″ / G ′) s = (tan δ) s is preferably less than 1 (more preferably 0.8 or less, Particularly preferably 0.5 or less).

When ω / 2π = 10 ⁻⁴ Hz (frequency at which the gel behaves as a liquid), (G ″ / G ′) L = (tan δ) _L is preferably 1 or more (more preferably 1. 5 or more, particularly preferably 2 or more).

The (tan [delta]) s and, (tan _[delta]) ratio of _{L {(tan δ) s /} (tan δ) L} is preferably less than 1 (more preferably 0.8 or less, particularly preferably 0 .5 or less).

<Measurement conditions>
Concentration of hydrogel-forming polymer in sustained-release composition: about 8% by mass
Temperature: Temperature about 10 ° C. higher than the sol-gel transition temperature of the sustained release composition Measuring instrument: Stress-controlled rheometer (Model name: AR500, manufactured by TA Instruments)

(Hydrogel-forming polymer)
The hydrogel-forming polymer that can be used in the sustained-release composition of the present invention is not particularly limited as long as it exhibits a thermoreversible sol-gel transition as described above (that is, has a sol-gel transition temperature). .

  Specific examples of the polymer in which the aqueous solution has a sol-gel transition temperature and reversibly shows a sol state at a temperature lower than the transition temperature include, for example, a block copolymer of polypropylene oxide and polyethylene oxide. Known polyalkylene oxide block copolymers; etherified celluloses such as methyl cellulose and hydroxypropyl cellulose; chitosan derivatives (KR Holme et al., Macromolecules, 24, 3828 (1991)) and the like are known.

  As a polyalkylene oxide block copolymer, a Pluronic F-127 (trade name, manufactured by BASF Wyandotte Chemicals Co.) gel in which polyethylene oxide is bonded to both ends of polypropylene oxide has been developed. It is known that this high-concentration aqueous solution of Pluronic F-127 becomes a hydrogel at about 20 ° C. or higher and becomes an aqueous solution at a lower temperature. However, in the case of this material, it becomes a gel state only at a high concentration of about 20% by mass or more, and even if it is kept at a high concentration of about 20% by mass or more and higher than the gelation temperature, water is added. The gel will dissolve. Pluronic F-127 has a relatively low molecular weight, and not only exhibits a very high osmotic pressure in a high gel state of about 20% by mass or more, but also easily penetrates the cell membrane. is there.

  On the other hand, in the case of etherified cellulose represented by methyl cellulose, hydroxypropyl cellulose and the like, usually, the sol-gel transition temperature is high and is about 45 ° C. or higher (N. Sarkar, J. Appl. Polym. Science, 24, 1073, 1979). In contrast, since the body temperature of a living body is usually around 37 ° C., the etherified cellulose is in a sol state, and it is practical to use the etherified cellulose as a sustained-release composition to be applied to the living body. Have difficulty.

  As described above, the problems of the conventional polymer in which the aqueous solution has a sol-gel transition point and reversibly shows a sol state at a temperature lower than the transition temperature are 1) higher than the sol-gel transition temperature. Once gelled, the gel dissolves when water is further added. 2) The sol-gel transition temperature is higher than the body temperature of the living body (around 37 ° C), and the body temperature is in the sol state. 3) In order to make it gel, it is necessary to make the polymer concentration of the aqueous solution very high.

  On the other hand, according to the study by the present inventors, in an embodiment in which the sustained release composition of the present invention is applied to a human body, the sol-gel transition temperature is preferably higher than 0 ° C. and not higher than 37 ° C. A hydrogel-forming polymer (for example, a plurality of blocks having a cloud point and a hydrophilic block are combined, and the aqueous solution has a sol-gel transition temperature and lower than the sol-gel transition temperature) It has been found that the above problem can be solved when a sustained-release composition is formed using a polymer that reversibly shows a sol state at a temperature.

(Suitable hydrogel-forming polymer)
The hydrogel-forming polymer utilizing a hydrophobic bond that can be suitably used as the sustained-release composition of the present invention is preferably formed by bonding a plurality of blocks having a cloud point and a hydrophilic block. The hydrophilic block is preferably present because the hydrogel becomes water-soluble at a temperature lower than the sol-gel transition temperature, and the plurality of blocks having a cloud point have a hydrogel having a sol-gel transition temperature. It is preferably present to change to a gel state at a higher temperature. In other words, a block having a cloud point dissolves in water at a temperature lower than the cloud point and changes to insoluble in water at a temperature higher than the cloud point, so that at a temperature higher than the cloud point, the block has a gel. It serves as a cross-linking point consisting of a hydrophobic bond to form. That is, the cloud point derived from the hydrophobic bond corresponds to the sol-gel transition temperature of the hydrogel.

  However, the cloud point and the sol-gel transition temperature do not necessarily match. This is because the cloud point of the “block having a cloud point” described above is generally affected by the bond between the block and the hydrophilic block.

  The hydrogel used in the present invention utilizes the property that not only the hydrophobic bond becomes stronger as the temperature increases, but also the change is reversible with respect to temperature. From the viewpoint that a plurality of crosslinking points are formed in one molecule and a gel having excellent stability is formed, the hydrogel-forming polymer preferably has a plurality of “blocks having cloud points”.

  On the other hand, the hydrophilic block in the hydrogel-forming polymer has a function of changing the water-soluble polymer to water-soluble at a temperature lower than the sol-gel transition temperature, as described above. It has a function of forming a hydrous gel state while preventing the hydrogel from aggregating and precipitating due to excessive increase in hydrophobic binding force at a temperature higher than the transition temperature.

  Furthermore, it is desirable that the hydrogel used in the present invention be decomposed and absorbed in vivo. That is, it is preferable that the hydrogel-forming polymer of the present invention is decomposed in a living body by a hydrolysis reaction or an enzymatic reaction to be absorbed and excreted as a low molecular weight body that is harmless to the living body.

  When the hydrogel-forming polymer of the present invention is formed by combining a plurality of blocks having a cloud point and a hydrophilic block, at least one of a block having a cloud point and a hydrophilic block is preferable. It is preferable that both are decomposed and absorbed in vivo.

(Multiple blocks with cloud points)
The block having a cloud point is preferably a polymer block having a negative solubility-temperature coefficient in water, and more specifically, polypropylene oxide, a copolymer of propylene oxide and another alkylene oxide, A polymer selected from the group consisting of poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers of N-substituted acrylamide derivatives and N-substituted methacrylamide derivatives, polyvinyl methyl ether, and polyvinyl alcohol partially acetylated products. Can be preferably used.

  In order to decompose and absorb a block having a cloud point in vivo, it is effective to make the block having a cloud point a polypeptide composed of a hydrophobic amino acid and a hydrophilic amino acid. Alternatively, a polyester-type biodegradable polymer such as polylactic acid or polyglycolic acid can be used as a block having a cloud point that is decomposed and absorbed in vivo.

  In an embodiment in which the sustained-release composition of the present invention is applied to a human living body, the above polymer (block having a cloud point) has a cloud point of higher than 4 ° C. and 40 ° C. or lower. A sol-gel transition temperature of a molecule (a compound in which a plurality of blocks having a cloud point and a hydrophilic block are bonded) is preferably from 0 ° C. to 37 ° C.

  Here, the cloud point is measured by, for example, cooling an aqueous solution of about 1% by mass of the above polymer (block having a cloud point) to form a transparent uniform solution, and then gradually increasing the temperature (temperature increase rate of about 1). C./min), and the point at which the solution becomes cloudy for the first time is taken as the cloud point.

  Specific examples of poly N-substituted acrylamide derivatives and poly N-substituted methacrylamide derivatives that can be used in the present invention are listed below.

  Poly-N-acroylpiperidine; poly-Nn-propylmethacrylamide; poly-N-isopropylacrylamide; poly-N, N-diethylacrylamide; poly-N-isopropylmethacrylamide; poly-N-cyclopropylacrylamide; Poly-N-acryloylpyrrolidine; poly-N, N-ethylmethylacrylamide; poly-N-cyclopropylmethacrylamide; poly-N-ethylacrylamide.

  The polymer may be a homopolymer or a copolymer of a monomer constituting the polymer and another monomer. As another monomer constituting such a copolymer, either a hydrophilic monomer or a hydrophobic monomer can be used. In general, copolymerization with a hydrophilic monomer raises the cloud point of the product and copolymerization with a hydrophobic monomer lowers the cloud point of the product. Therefore, a polymer having a desired cloud point (for example, a cloud point higher than 4 ° C. and lower than 40 ° C.) can be obtained also by selecting the monomer to be copolymerized.

(Hydrophilic monomer)
Examples of the hydrophilic monomer include N-vinyl pyrrolidone, vinyl pyridine, acrylamide, methacrylamide, N-methyl acrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, and acrylic having an acid group. Acid, methacrylic acid and salts thereof, vinyl sulfonic acid, styrene sulfonic acid and the like, and N, N-dimethylaminoethyl methacrylate, N, N-diethylaminoethyl methacrylate, N, N-dimethylaminopropyl having a basic group Examples include, but are not limited to, acrylamide and salts thereof.

(Hydrophobic monomer)
On the other hand, examples of the hydrophobic monomer include acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate and glycidyl methacrylate, N-substituted alkylmethacrylamide derivatives such as Nn-butylmethacrylamide, vinyl chloride, acrylonitrile and styrene. And vinyl acetate and the like, but are not limited thereto.

(Hydrophilic block)
On the other hand, as the hydrophilic block to be combined with the above-described block having a cloud point, specifically, methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide, polymethacrylamide , Poly N-methylacrylamide, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid and their salts; poly N, N-dimethylaminoethyl methacrylate, poly N, N-diethylaminoethyl methacrylate , Poly N, N-dimethylaminopropylacrylamide and salts thereof.

  The hydrophilic block is desirably decomposed, metabolized and excreted in vivo, and hydrophilic biopolymers such as proteins such as albumin and gelatin, and polysaccharides such as hyaluronic acid, heparin, chitin and chitosan are preferably used. .

  The method for bonding the block having a cloud point and the hydrophilic block is not particularly limited. For example, a polymerizable functional group (for example, acryloyl group) is introduced into one of the blocks to give the other block. This can be done by copolymerizing monomers. Further, the combined product of a block having a cloud point and the hydrophilic block may be obtained by block copolymerization of a monomer that gives a block having a cloud point and a monomer that gives a hydrophilic block. Is possible. In addition, the block having a cloud point and the hydrophilic block are bonded to each other by introducing a reactive functional group (for example, a hydroxyl group, an amino group, a carboxyl group, an isocyanate group, etc.) in advance and bonding them together by a chemical reaction. Can also be done. At this time, usually a plurality of reactive functional groups are introduced into the hydrophilic block. In addition, the bond between the polypropylene oxide having a cloud point and the hydrophilic block is repeated, for example, by anionic polymerization or cationic polymerization by repeatedly repeating propylene oxide and a monomer (for example, ethylene oxide) constituting “another hydrophilic block”. By polymerizing, a block copolymer in which polypropylene oxide and a “hydrophilic block” (for example, polyethylene oxide) are bonded can be obtained. Such a block copolymer can also be obtained by copolymerizing monomers constituting a hydrophilic block after introducing a polymerizable group (for example, acryloyl group) to the terminal of polypropylene oxide. Furthermore, the polymer used in the present invention can also be obtained by introducing a functional group capable of binding reaction with a functional group (for example, hydroxyl group) at the end of polypropylene oxide into the hydrophilic block and reacting both. . Moreover, the hydrogel-forming polymer used in the present invention can also be obtained by linking materials such as Pluronic F-127 (trade name, manufactured by Asahi Denka Kogyo Co., Ltd.) in which polyethylene glycol is bonded to both ends of polypropylene glycol. Can be obtained.

  The polymer of the present invention in a mode including a block having a cloud point is water-soluble together with the hydrophilic block at the temperature lower than the cloud point, because the above-mentioned “block having a cloud point” is present in the molecule. It is completely dissolved in water and shows a sol state. However, when the temperature of the polymer aqueous solution is raised to a temperature higher than the above cloud point, the “block having a cloud point” present in the molecule becomes hydrophobic, and is associated between separate molecules by hydrophobic interaction. To do.

  On the other hand, since the hydrophilic block is still water-soluble at this time (when heated to a temperature higher than the cloud point), the polymer of the present invention is a hydrophobic association part between blocks having a cloud point in water. A hydrogel having a three-dimensional network structure with a cross-linking point as a cross-linking point is generated. When the temperature of this hydrogel is cooled again to a temperature lower than the cloud point of the “block having cloud point” existing in the molecule, the block having the cloud point becomes water-soluble and the crosslinking point due to hydrophobic association is released. The hydrogel structure disappears and the polymer of the present invention becomes a complete aqueous solution again. Thus, the sol-gel transition of the polymer of the present invention in a preferred embodiment is based on reversible changes in hydrophilicity and hydrophobicity at the cloud point of the block having the cloud point present in the molecule. Therefore, it has complete reversibility in response to temperature changes.

(Gel solubility)
As described above, the hydrogel-forming polymer of the present invention containing at least a polymer having a sol-gel transition temperature in an aqueous solution is substantially insoluble in water at a temperature higher than the sol-gel transition temperature (d ° C.). It is reversibly water soluble at a temperature (e ° C.) lower than the sol-gel transition temperature.

  The high temperature (d ° C.) is preferably a temperature that is 1 ° C. or more higher than the sol-gel transition temperature, and more preferably 2 ° C. or more (particularly 5 ° C. or more). The “substantially water-insoluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (d ° C.) is 5.0 g or less (more preferably 0.5 g or less, particularly 0.1 g or less). ) Is preferable.

  On the other hand, the above-mentioned low temperature (e ° C.) is preferably 1 ° C. or more lower than the sol-gel transition temperature (in absolute value), more preferably 2 ° C. or higher (particularly 5 ° C. or higher). preferable. The “water-soluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (e ° C.) is preferably 0.5 g or more (more preferably 1.0 g or more). Further, “reversibly water-soluble” means that the aqueous solution of the hydrogel-forming polymer is once gelled (at a temperature higher than the sol-gel transition temperature), even after the sol-gel transition temperature. It means that it exhibits the above-mentioned water solubility at a low temperature.

  The polymer preferably has a viscosity of 10 to 3,000 centipoise (more preferably 50 to 1,000 centipoise) when its 10% aqueous solution is 5 ° C. Such viscosity is preferably measured under the following measurement conditions, for example.

Viscometer: Stress-controlled rheometer (Model name: AR500, manufactured by TA Instruments)
Rotor diameter: 60mm
Rotor shape: parallel plate

  Even if the aqueous solution of the hydrogel-forming polymer of the present invention is gelled at a temperature higher than the sol-gel transition temperature and then immersed in a large amount of water, the gel is not substantially dissolved. The said characteristic of the said sustained release composition can be confirmed as follows, for example.

  That is, 0.15 g of the hydrogel-forming polymer of the present invention is dissolved in 1.35 g of distilled water at a temperature lower than the sol-gel transition temperature (for example, under ice cooling) to prepare a 10 wt% aqueous solution. The aqueous solution was poured into a plastic petri dish having a diameter of 35 mm and heated to 37 ° C. to form a gel having a thickness of about 1.5 mm in the petri dish, and then the weight of the whole petri dish containing the gel (F gram) is measured. Subsequently, after the whole petri dish containing the gel was allowed to stand at 37 ° C. for 10 hours in 250 ml of water, the weight (g gram) of the whole petri dish containing the gel was measured, and the dissolution of the gel from the gel surface was measured. Evaluate presence or absence. At this time, in the hydrogel-forming polymer of the present invention, the weight reduction rate of the gel, that is, (f−g) / f is preferably 5.0% or less, and more preferably 1.0%. Or less (particularly 0.1% or less).

  The aqueous solution of the hydrogel-forming polymer of the present invention is gelled at a temperature higher than the sol-gel transition temperature, and then immersed in a large amount of water (by volume, about 0.1 to 100 times the gel). However, the gel does not dissolve over a long period of time. Such a property of the polymer used in the present invention is achieved by, for example, the presence of two or more (plural) blocks having a cloud point in the polymer.

  On the other hand, when a similar gel was prepared using the aforementioned Pluronic F-127 in which polyethylene oxide was bonded to both ends of polypropylene oxide, the gel was completely dissolved in water after standing for several hours. The inventors have found that this is the case.

  From the standpoint of suppressing the non-gelling cytotoxicity to the lowest possible level, the concentration in water, that is, {(polymer) / (polymer + water)} × 100 (%), 20% or less (and 15 It is preferable to use a hydrogel-forming polymer that can be gelled at a concentration of not more than%, particularly not more than 10%.

  The molecular weight of the hydrogel-forming polymer used in the present invention is preferably from 30,000 to 30 million, more preferably from 100,000 to 10 million, and still more preferably from 500,000 to 5 million.

(Active substance)
In the present invention, the fine particles as one component constituting the sustained release composition are fine particles containing at least an active substance. From the viewpoint of sustained release control, the fine particles are preferably fine particles capable of exhibiting sustained release of the active substance by themselves. Here, the “active substance” refers to a substance for which sustained release itself is beneficial, and that the release from the fine particles can be detected by some means. A physiologically active substance having a physiologically active action is an embodiment of this “active substance”.

  In the present invention, “fine particles capable of exhibiting sustained release of an active substance” are not particularly limited. That is, it can be appropriately selected from known fine particles (liposomes, dendrimers, fat preparations, microcapsules, microspheres, polymer micelles, etc.) and used as “fine particles” in the present invention. Regarding the composition, production method, and usage of such “fine particles capable of exhibiting sustained release of active substances”, supervised by Minoru Koishi, “Development and application of micro / nano-based capsules / fine particles” as necessary In 2003, reference can be made to CMC Publishing Co., Ltd. Regarding sustained release of physiologically active substances, Kohei Miyao, Actual Drug Delivery System, 1986, Medicinal Journal can be referred to as necessary.

  As long as the sustained release of the active substance can be expressed, the form of supporting or holding the active substance on the fine particles is not particularly limited. For example, the active substance may be encapsulated in the fine particles, or may be adhered, adsorbed or bonded to the surface of the fine particles.

(Bioactive substance)
The physiologically active substance in the present invention is a general term for organic substances and inorganic substances that are involved in and affect minute life phenomena of living organisms. The physiologically active substance is not particularly limited as long as it is an arbitrary compound or substance composition that can be administered to animals, preferably humans. For example, the active substance includes a compound or composition that exhibits physiological activity in the body and is effective in preventing or treating a disease, such as a compound or composition used for diagnosis of a contrast agent, a gene useful for gene therapy, and the like. included.

(Specific examples of active substances)
Various pharmaceuticals can be suitably used as the active substance (or physiologically active substance) in the present invention. For example, anticancer agents, antibiotics, analgesics, immunopotentiators, immunosuppressants, antithrombotic agents, bronchodilators, hypertensive agents, growth factors, hormones, and the like can be used.

  As the active substance in the present invention, for example, an antitumor agent (anticancer agent) can be suitably used. Examples of antitumor agents include alkylating agents, various antimetabolites, antitumor antibiotics, other antitumor agents, antitumor plant components, BRM (biological response regulator), angiogenesis inhibitors, cell adhesion Inhibitors, matrix metalloprotease inhibitors, hormones, and the like.

(Alkylating agent)
More specifically, examples of the alkylating agent include chloroethylamine alkylating agents such as nitrogen mustard, nitrogen mustard N-oxide, ifosfamide, melphalan, cyclophosphamide, and chlorambucil; for example, carbocon, thiotepa Aziridine-based alkylating agents such as dibromomannitol and dibromodulcitol; for example, nitrosourea-based alkylating such as carmustine, lomustine, semustine, nimustine hydrochloride, chlorozotocin and ranimustine Agents; sulfonic acid esters such as busulfan, improsulfan tosylate and pipersulfan; dacarbazine; procarbazine and the like.

(Antimetabolite)
Examples of various antimetabolites include purine antimetabolites such as 6-mercaptopurine, azathioprine, 6-thioguanine, and thioinosine; fluorouracil (5-fluorouracil), tegafur, tegafur uracil, carmofur, doxyfluridine, broxuridine, cytarabine And pyrimidine antimetabolites such as enocitabine; antifolate antimetabolites such as methotrexate and trimetrexate, and salts or complexes thereof.

(Anti-tumor agent)
Antitumor antibiotics include, for example, anthracyclines such as daunorubicin, aclacinomycin A (acralubicin), ansamitocin, doxorubicin (doxorubicin hydrochloride), pirarubicin, epirubicin; actinomycins such as actinomycin D; chromomycin a _3, etc. chromomycin system; mitomycin system mitomycin C and the like; bleomycin, bleomycin system such as peplomycin; and their salts or complexes thereof.

  Examples of other antitumor agents include taxol (paclitaxel), taxotere (docetaxel), batimastat, cisplatin, carboplatin, tamoxifen, L-asparaginase, acebraton, schizophyllan, picibanil, ubenimex, krestin, and the like, or a salt or complex thereof. The body is mentioned.

  Examples of the antitumor plant component include plant alkaloids such as camptothecin, vindesine, vincristine and vinblastine; epipodophyllotoxins such as etoposide and teniposide; and salts or complexes thereof. In addition, examples include pipbloman, neocartinostatin, and hydroxyurea.

(BRM)
Examples of the BRM include tumor necrosis factor, indomethacin, and salts or complexes thereof. Examples of the angiogenesis inhibitor include fumagillol derivatives and salts or complexes thereof. Examples of the cell adhesion inhibitor include substances having an RGD sequence, and salts or complexes thereof. Examples of matrix metalloprotease inhibitors include marimastat, batimastat and the like, and salts or complexes thereof. Examples of hormones include hydrocortisone, dexamethasone, methylprednisolone, prednisolone, plasterone, betamethasone, triamcinolone, oxymetholone, nandrolone, methenolone, phosfestol, ethinylestradiol, chlormadinone, medroxyprogesterone, and salts or complexes thereof. Can be mentioned.

(Antibiotics)
As the active substance in the present invention, for example, an antibiotic can be preferably used. Specific examples include penicillin, streptomycin, chloramphenicol, chlortetracycline, oxytetracycline, cephalosporin, kanamycin, dibekacin, erythromycin, clindamycin, griseofulvin, gentamicin, actinomycin, aclarubicin, ampicillin, spectinomycin , Tobramycin, bleomycin, amikacin, sisomycin, fradiomycin (neomycin), baromomycin, cycloserine (oxazamycin), rifampicin, biomycin, daunomycin, nystatin, tricomycin, polymyxin, colistin, bacitracin, leucomycin, josamycin, spiramycin, gramicidin, Fosfomycin, novobiocin, lincomey It down, nystatin, trichomycin, amphotericin B, pimaricin, preventor mycin, Azzaro mycin, Bariochin, Pirorunitorin, griseofulvin, and the like Zarukomaishin.

(Painkiller)
As the active substance in the present invention, for example, an analgesic can be suitably used. Specific examples of narcotic analgesics (central analgesics) include opium, morphine, codeine, morphine, oxycodone, pentazocine, dihydrocodeine, pethidine, methadone and the like as antipyretic analgesics (anti-inflammatory agents) Sodium salicylate, acetylsalicylic acid (aspirin), salicylamide, acetaminophen, phenacetin, antipyrine, aminopyrine, sulpyrine, bromelsin, lysozyme, proctase, glycyrrhizic acid, glycyrrhetinic acid, mefenamic acid, phenylbutazone, indomethacin, ibuprofen, loxoprofen, ketoprofen , Allantoin, guaiazulene and their derivatives and their salts, ε-aminocaproic acid, zinc oxide, diclofenac sodium, aloe extract, salvia extraction , Arnica extract, chamomile extract, white birch extract, Hypericum extract, and the like eucalyptus extract and Sapindus extract.

(Immune enhancer)
As the active substance in the present invention, for example, an immunopotentiator can be suitably used. Such materials include any antigen, hapten, organic moiety, or organic or inorganic compound that can generate an immune response and be combined into microparticles. For example, the supported materials include immunoglobulins, antibodies including monoclonal antibodies and non-iodotype antibodies, antibody fragments, interleukins, interferons, viruses, viral fragments and other genetic materials. More specifically, production of vaccines against malaria (US Pat. No. 4,735,799), cholera (US Pat. No. 4,751,064) and urinary tract infection (US Pat. No. 4,740,585). Synthetic peptides that can be used in bacteria, bacterial polysaccharides for the production of bactericidal vaccines (US Pat. No. 4.695.624) and viral proteins for the production of antiviral vaccines for the prevention of diseases such as AIDS and hepatitis Alternatively, virus particles can be used.

(Immunosuppressant)
As the active substance in the present invention, for example, an immunosuppressive agent can be suitably used. Specific examples include sirolimus, everolimus, tacrolimus, methotrexate, cyclophosphamide, azathioprine, and mizoribine.

(Antiviral agent)
As the active substance in the present invention, for example, an antiviral agent can be preferably used. Specific examples include acyclovir, zidovudin, and interferons.

(Cell growth factor, etc.)
As the active substance in the present invention, for example, cell growth factors, hormones or local chemical mediators can be preferably used. Specific examples include fibroblast growth factor (FGF), epithelial growth factor (EGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor. (Hepatocyte growth factor, HGF), platelet derived growth factor (PDGF) and other cell growth factors, insulin, somatotropin, somatomedin, corticotropin (ACTH), parathyroid hormone (PTH), thyroid stimulating hormone (TSH) and other proteins or glycoproteins, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin sulfate, heparin, keratan sulfate, and other mucopolysaccharides and salts thereof, amino acids derivatives such as TSH release factor, vasopressin, somatostatin, interleukins , Interfero , Tumor necrosis factor, granulocyte colony-stimulating factor, cortisol, estradiol, testosterone, thrombomodulin, 17β-estradiol, norethindrone, prednisolone, dexamethasone, betamethasone and the like. Examples of local chemical mediators include proteins such as nerve cell growth factors, peptides such as chemotactic factors, amino acid derivatives such as histamine, and fatty acid derivatives such as prostaglandins.

(Antithrombotic agent)
As the active substance in the present invention, for example, an antithrombotic agent can be preferably used. Examples of the antithrombotic agent include heparin, warfarin, acenocoumarol, phenindione, EDTA and the like.

(Bone formation promoting agent)
As the active substance in the present invention, for example, a substance having a cartilage formation promoting action or a bone formation promoting action can be suitably used. For example, calcium agent, active vitamin D ₃ (eg, 1α-hydroxyvitamin D ₃ , 1α-2,5-dihydroxyvitamin D ₃ , floccitriol, secalciferol, etc.), calcitonin and its derivatives, peptides, ( Interleukin-1β converting enzyme, cathepsin B, cathepsin L and the like, non-peptide chondrogenesis promoting substances, substances having a cartilage promoting action or osteogenesis promoting action such as benzothiopyran and benzothiebin derivatives (for example, JP-A-3-232880) Compounds, salts thereof), β-alanyl-3,4-dihydroxyphenylalanine, xanthine derivatives, polyphenols, and the like described in JP-A-4-364179, JP-A-8-231369, JP-A-2000-72678, etc. Compounds, prostaglandins, Anti-rheumatic drugs such as sodium thiomalate, auranofin, D-penicillamine, bucillamine, lobenzalit, actarit, salazosulfapyridine, anti-rheological agents such as aminoglycoside, cephalosporin, tetracycline, anti-polyene antibiotics, imidazole, triazole, etc. Fungal agents, sterols such as cholesterol, collagen, elastin, keratin and derivatives thereof and salts thereof, for example, carbohydrates such as sugar and starch, cell receptor proteins, enzymes, neurotransmitters, glycoproteins and the like can be used.

(Tyrosinase activity inhibitor)
As the active substance in the present invention, for example, a tyrosinase activity inhibitor can be suitably used. For example, cysteine and its derivatives and salts thereof, Sempukuka extract, Caiket extract, Sampens extract, Sakuhakuhi extract, Toki extract, Ibukitorano extract, Clara extract, Hawthorn extract, Silly extract, Hop extract , Neubara extract and Yokuinin extract.

(Carotenoid derivative)
As the active substance in the present invention, for example, carotenoids and derivatives thereof can be suitably used. Examples include esters of astaxanthin, for example, amino acid esters such as glycine and alanine, carboxylic acid esters such as acetates and citrates and salts thereof, inorganic salt esters such as phosphates and sulfates, and salts thereof, Glycosides such as glucosides, highly unsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, unsaturated fatty acids such as oleic acid and linoleic acid, and fatty acid esters selected from saturated fatty acids such as palmitic acid and stearic acid, etc. And monoesters selected from the above or the same or different diesters.

(Toxins)
For example, toxins can be suitably used as the active substance in the present invention. For example, diphtheria toxin, gelonin, exotoxin A, abrin, modesin, ricin or toxic fragments thereof can be used.

(Metal or metal ion)
As the active substance in the present invention, for example, a metal or a metal ion can be suitably used. For example, Group VIIIA (Fe, Co, Ni, Ru, Rh, Pd, Os, Ir, Pt), Group IVB (Pb, Sn, Ge), Group IIA (SC, Y, lanthanides, and actinides) Group IIIB (B, Al, Ga, In, Tl), Group IA alkali metals (Li, Na, K, Rb, Cs, Fr), and Group IIA alkaline earth metals (Be, Mg, Metals such as Ca, Sr, Ba, Ra) and transition metals and their metal ions can be used.

(Radionuclide)
As the active substance in the present invention, for example, a radionuclide can be suitably used. For example, from actinides or lanthanides, or other similar transition elements, or other elements such as ⁴⁷ Sc, ⁶⁷ Cu, ⁶⁷ Ga, ⁸² Rb, ⁸⁹ Sr, ⁸⁸ Y, ⁹⁰ Y, ^99m Tc, ¹⁰⁵ Rh, ^{109 Pd, 111 In, 115m In} , 125 I, 131 I, 140 Ba, 140 La, 149 Pm, 153 Sm, 159 Gd, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 194 Ir and ¹⁹⁹ Au Preferably, those generated from ⁸⁸ Y, ⁹⁰ Y, ^99m Tc, ¹²⁵ I, ¹³¹ I, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ⁶⁷ Ga, ¹¹¹ In, ^{115 m} In and ¹⁴⁰ La are used. it can.

(Fragrance)
For example, a fragrance can be suitably used as the active substance in the present invention. As the fragrance, any of animal-based and plant-based natural fragrances, synthetic fragrances, and blended fragrances may be used. In the present invention, since the fine particles containing a fragrance are supported on the hydrogel of the present invention to control the release characteristics of the fragrance, the fragrance is preferably water-insoluble. Here, that the fragrance is water-insoluble means that the solubility of the fragrance in water is 1% or less, preferably 0.5% or less, more preferably 0.1% or less at 25 ° C.

(Fine particles containing active substance)
In the present invention, known microparticles containing an active substance such as liposomes, dendrimers, microspheres, fat preparations, lipid microspheres (lipospheres), microcapsules, and polymer micelles are used as the microparticles containing the active substance. be able to.

  Furthermore, blood cells such as platelets, white blood cells, red blood cells, and lymphocytes, cancer cells, and various normal cells can also be used as fine particles containing an active substance. For example, if platelets (microparticles) are used as the sustained release composition of the present invention, PDGF (active substance) contained in the platelets can be sustainedly released. Alternatively, if insulin-producing cells (microparticles) are used as the sustained-release composition of the present invention, insulin (active substance) can be sustained-released.

(Particle size)
In the present invention, the particle size of the fine particles containing the active substance is not particularly limited as long as it can be retained in the hydrogel for a desired time (for example, retained by dispersion in the hydrogel) until sustained release. . From the viewpoint of easy retention in the hydrogel, the particle diameter of the fine particles containing the active substance is preferably in the range of 1 nm to 1 mm, more preferably in the range of 10 nm to 0.1 mm (particularly 20 nm to 20 μm). Is preferable. When the size of the fine particles is below this range, the diffusion rate of the fine particles in the hydrogel increases, and it tends to be difficult to hold the active substance in the sustained-release composition of the present invention over a long period of time. Become. On the other hand, if the size of the fine particles exceeds this range, it tends to be difficult to uniformly distribute the fine particles in the sustained release composition of the present invention.

  When the above-mentioned microparticles containing a physiologically active substance are used for DDS (Kohei Miyao, Drug Delivery System, 1986, Pharmaceutical Journal, Inc.) is administered to a living body by injection. Since it easily circulates throughout the body, it is difficult to slow release the active substance locally at the target site. On the other hand, since the sustained-release composition of the present invention is such that fine particles containing an active substance are administered in a liquid state and then gelled at body temperature, the fine particles containing an active substance can be placed locally at the administration site. It is possible to release the active substance at a site locally.

(Liposome)
The liposome that can be used in the present invention is a spherical closed vesicle composed of a phospholipid bilayer membrane in an aqueous phase, and its particle size can be adjusted from 20 nm to 20 μm in terms of volume average particle size. The volume average particle diameter can be determined by a dynamic light scattering method. Small active lamellar vesicles (SUV) and large unilamellar vesicles (LUV) composed of a single bilayer film, and multilamellar vesicles (MLV) composed of a plurality of bilayer films, all of which are active substances of the present invention It can be used as fine particles containing.

  Phospholipids that form liposomes are phosphatidylcholine (lecithin), phosphatidylserine, sphingomyelin, and the like. Usually, lecithin is used, and neutral lipid such as cholesterol is added to stabilize the membrane. In addition, for the purpose of improving the encapsulation efficiency of the active substance in the liposome or imparting organ directivity to the liposome, a lipid having a charge or a lipid combined with a hydrophilic polymer can be added. As the lipid to be positively charged, aliphatic amines such as stearylamine can be used, and as the lipid to be negatively charged, fatty acids such as stearic acid and myristic acid, phosphatidic acid, phosphatidylglycerol, dicetyl phosphate and the like can be used.

  The addition of lipids bound with hydrophilic polymers is mainly aimed at the surface modification of liposomes. By binding a monoclonal antibody to the liposome surface, the affinity of the liposome for a specific antigen can be increased. In addition, a lipid to which a hydrophilic polymer such as a polysaccharide or polyethylene glycol (PEG) is bound can be mixed with a lipid forming a liposome to be exposed on the surface of the liposome. Modification of the liposome surface with PEG suppresses protein adsorption to the liposome surface, prevents aggregation of the liposome in the plasma, avoids liposome trapping by the intraretinal system, and extends the residence time in the circulating blood. (H. Yoshioka, Biometerials, 12, 861 (1991).

  It does not specifically limit as a method of manufacturing a liposome, You may use a well-known method. For example, liposomes can be produced by using the above phospholipid and aqueous phase by the thin film method, reverse phase evaporation method, ethanol injection method, ether injection method, dehydration-rehydration method and the like. Of these, the ether injection method is preferred. Volume average particle size can be adjusted by methods such as ultrasonic irradiation, ultrasonic irradiation after freezing and thawing, extrusion method, French press method, and homogenization method (DDLasic, “Liposomes: from basic to applications ", Elsevier Science Publishers, p. 1-171 (1993)). Here, the aqueous phase may be an aqueous solution containing water and an aqueous solution and constituting the inside of the liposome. There is no particular limitation as long as it is usually used in this technical field, but a sodium chloride aqueous solution, a buffer solution such as a phosphate buffer solution or an acetate buffer solution, a glucose aqueous solution, a sugar aqueous solution such as trehalose, or a mixed aqueous solution thereof is preferable. It is. In general, in order to keep the structure of liposomes administered in vivo stable, the aqueous phase used for the production of liposomes is outside the liposomes, that is, isotonic with respect to body fluids, and the osmotic pressure applied to the inside and outside of the liposomes is small. It is preferable.

  In order to contain the active substance in the liposome, it may be dissolved or mixed in the aqueous phase or phospholipid during the production process. The water-soluble active substance can be retained in the inner aqueous layer of the liposome, and the fat-soluble active substance can be retained in the phospholipid bilayer membrane of the liposome.

(Dendrimer)
Dendrimer is a compound form derived from dendra, which means tree in Greek, and has a structure in which multiple branched molecular chains extend radially from the center of the molecule. Because of its branched structure, the spatial extent of the dendrimer compound is relatively small relative to the molecular weight, and usually has a substantially spherical size up to several tens of nm in diameter. Compared to conventional linear polymer compounds, dendrimer compounds allow independent molecular design of core, branched chain, surface, etc., and can achieve three-dimensional molecular construction. A significant improvement in the function of the compound can be expected by effectively spatially arranging specific atomic groups according to the application. Its application is expected in a wide range of fields such as nanocapsules and gene vectors.

  Branched polymer compounds such as polyethyleneimine and polypropyleneimine produced industrially are also one type of dendrimer compound, and these are formed by a polymerization reaction of a reactive monomer in which branching is naturally promoted. There are two methods for synthesizing dendrimer compounds based on molecular design for the expression of the desired function: the divergent method and the convergent method. The Divergent method is a production method in which step reactions are repeated on the starting material to increase the number of branches. On the other hand, the Convergent method is a production method in which dendrons are synthesized stepwise from the peripheral components and finally a plurality of dendrons are combined.

  In order to make the dendrimer contain an active substance, a conventionally known method (for example, the method disclosed in JP-T-08-510761 can be used).

(Microsphere)
In general, microspheres are used as a microsphere (1 to several tens of microns) matrix DDS mainly for sustained release of drugs and a certain degree of targeting. Furthermore, a stronger targeting function can be imparted by binding an antibody or providing magnetism. As the component, biopolymers such as albumin and collagen, or synthetic polymers can be used.

(Liposphere)
Although it is a kind of microsphere, a micro droplet microsphere of oil and fat is called a lipid microsphere (liposphere) and can be suitably used in the present invention. For example, an intralipid lipid injection can be used as the fine particles of the present invention. Various lipophilic active substances can be supported on the liposphere.

(Micro capsule)
The microcapsules that can be used in the present invention are those in which fine particles such as solid, liquid, solution or suspension or fine droplets are coated with a uniform film of polymer. Microspheres are homogeneous throughout, whereas microcapsules have different septa and contents. The coating material is selected from natural and synthetic film-forming polymers, such as carboxymethyl cellulose, cellulose acetate phthalate, ethyl cellulose, gelatin, gelatin-gum arabic, nitrocellulose, polyvinyl alcohol, propylhydroxycellulose, shellac, succinylate. Gelatin, wax and the like can be used.

  Microcapsule preparation methods include coacervation method (phase separation method), interfacial polymerization method, air suspension method, orifice method (porous centrifuge method), electrostatic deposition method, spray drying method, pan coating method, etc. Can be used (Kohei Miyao, Drug Delivery System, 1986, Pharmaceutical Journal).

(Polymer micelle)
As the fine particles containing an active substance that can be used in the present invention, polymer micelles in which a poorly water-soluble drug is encapsulated using a block copolymer having a hydrophilic segment and a hydrophobic segment (for example, see Japanese Patent No. 2777530) are preferably used. Can be used.

  As a method for preparing the polymer micelle, for example, the following method described in the above publication can be adopted.

  a) A poorly water-soluble drug is dissolved in a water-miscible organic solvent, if necessary, and stirred and mixed with an aqueous block copolymer dispersion. In some cases, entrapment of the drug in the polymer micelle can be promoted by heating during stirring and mixing.

  b) A water-immiscible organic solvent solution of a poorly water-soluble drug is mixed with a block copolymer-dispersed aqueous solution, and the organic solvent is volatilized while stirring.

  c) After dissolving a poorly water-soluble drug and a block copolymer in a water-miscible organic solvent, the resulting solution is dialyzed against a buffer and / or water using a dialysis membrane.

Alternatively, the following method described in JP-A-2001-226294 can be employed.
d) Dissolve poorly water-soluble drug and block copolymer in water-immiscible organic solvent, mix the resulting solution with water, stir to form an oil-in-water (O / W) type emulsion, then strip the organic solvent Let

  Furthermore, polymer micelles encapsulating a poorly water-soluble drug can be freeze-dried by adding saccharides, inorganic salts, polyethylene glycol and the like as auxiliary agents by the method described in JP-A-2003-26812. This freeze-dried product is easily re-dispersed in water to easily reconstitute a polymer micelle dispersion in which a poorly water-soluble drug is encapsulated.

(Suitable block copolymer)
The block copolymer that can be suitably used in the present invention is a so-called AB-type or ABA-type block copolymer comprising a hydrophilic segment (hereinafter also referred to as A segment) and a hydrophobic segment (hereinafter also referred to as B segment). be able to. Examples of the polymer constituting the A segment include, but are not limited to, polyethylene glycol (or polyoxyethylene), polysaccharide, polyvinyl pyrrolidone, and polyvinyl alcohol. Of these, preferred are those composed of polyethylene glycol. Although not limited, it is more preferable that the polyethylene glycol segment has 10 to 2500 repeating units of oxyethylene.

  The A segment may be any low molecular functional group or part of a molecule (eg, lower alkyl, amino group, carboxyl, etc.) as long as it does not adversely affect the formation of polymer micelles at the end opposite to the bond end with the B segment. Group, sugar residue, protein residue, etc.).

  On the other hand, the B segment is not limited, but includes polyamino acid esters (polyaspartic acid esters, polyglutamic acid esters, and partial hydrolysates thereof), poly (meth) acrylic acid esters, polylactides, polyesters, and the like. Can give. The B segment is the same as that described for the A segment, as long as it does not adversely affect the interaction between the drug and the B segment when forming a polymer micelle at the end opposite to the binding end with the A segment. It can have a low molecular functional group. Typical examples of such a block copolymer include polymers described in, for example, Japanese Patent No. 2777530, WO96 / 32434, WO96 / 33233, and WO97 / 06202, and those derived from them. be able to.

(Diffusion rate of fine particles)
In an embodiment in which the sustained-release composition of the present invention is applied to a human living body, the diffusion coefficient of fine particles containing an active substance in the sustained-release composition is 1 × 10 ⁻⁸ (cm ² / sec) at 37 ° C. Hereinafter, it is more preferably 1 × 10 ⁻⁹ (cm ² / sec) or less, and further preferably 1 × 10 ⁻¹⁰ (cm ² / sec) or less. In the present invention, the phrase “fine particles do not substantially diffuse” means that the diffusion coefficient of the fine particles is 1 × 10 ⁻¹⁰ (cm ² / sec) or less.

The diffusion coefficient of the fine particles in the hydrogel can be determined by the “early-time” approximation method described in the literature (Eric KLLee et al., Journal of Membrane Science, 24, 125-143 (1985)). In this method, the process in which fine particles uniformly dispersed in a hydrogel plate having a uniform thickness L (cm) elute from both surfaces of the hydrogel plate is observed over time. If the elution amount of fine particles at time t (sec) is Mt, and the elution amount after infinite time is M∞, the diffusion coefficient D (cm ² / The following equation (1) holds for (sec).

Mt / M∞ = (Dt / π) ^1/2 × 4 / L (1)
Accordingly, the diffusion coefficient D can be calculated from the slope of a straight line plotting the elution rate up to the time t with respect to the square root of the elapsed time t.

(Active substance controlled release mechanism)
The release of the active substance from the sustained-release composition of the present invention is as follows: 1) diffusion process of active substance in hydrogel, 2) diffusion process of fine particles containing active substance in hydrogel, 3) fine particles containing active substance It is controlled by a combination of four kinds of release processes, that is, active substance release process and 4) hydrogel disintegration process.

  The present inventors have already proposed 1) a method for controlling the diffusion process of an active substance in a hydrogel (Japanese Patent Application Laid-Open No. 2004-043749). Also in the present invention, when the active substance is released from the fine particles in the hydrogel, the released active substance is released from the sustained release composition of the present invention through a process of diffusing in the hydrogel. become.

  Further, the active substance alone may be mixed in the sustained-release composition containing the fine particles containing the active substance of the present invention without previously supporting the active substance on the fine particles. At this time, the active substance supported on the fine particles and the active substance not supported may be the same or different active substances. In this case, the active substance normally not supported on the fine particles is released relatively early, and then the active substance supported on the fine particles is released gradually. Therefore, the amount of active substance released can be controlled in two stages, the initial stage and the latter stage, or different active substances can be caused to act sequentially.

  In the present invention, 2) a more advanced controlled release of the active substance can be achieved by combining the diffusion process of the fine particles containing the active substance in the hydrogel. Specifically, in an embodiment in which the composition of the present invention is applied to a human body, the diffusion rate of the fine particles can be reduced by increasing the particle size of the fine particles containing the active substance. Alternatively, by increasing the concentration of the hydrogel-forming polymer, the hydrogel network structure at a living body temperature (37 ° C.) can be densified to reduce the diffusion rate of the fine particles containing the active substance.

  Furthermore, in the present invention, 3) an active substance release process from fine particles containing an active substance can be combined. In general, fine particles containing an active substance such as a liposome preparation are very stable outside the living body, and the active substance is hardly released from the fine particles during storage. However, in the living body, fine particles are decomposed by the action of an enzyme or the like, and the contained active substance is released to exert its physiologically active function. In addition, in the hydrogel of the present invention, there are cases where normally stable fine particles release an active substance contained by interaction with the hydrogel-forming polymer. This is because the hydrogel-forming polymer of the present invention is composed of a polymer block having a relatively high hydrophobicity (polymer block having a cloud point).

  Fine particles such as liposomes, polymer micelles, and lipid spheres that can be used in the present invention have a fine particle structure formed mainly by hydrophobic interaction. Therefore, the fine particle structure is affected by the hydrophobic interaction with the hydrogel-forming polymer of the present invention having a relatively highly hydrophobic polymer block, and the active substance contained in the fine particles is gradually released from the fine particles. It is considered to be done.

  For example, when multilamellar liposomes are used as fine particles containing an active substance, the phospholipid bilayer membrane of the liposomes is affected in order from the outermost layer in contact with the hydrogel-forming polymer to the inner layer side. Therefore, since the active substance contained in the outermost layer is released first, and the active substance contained in the inner layer side is gradually released, it is possible to design a highly controlled sustained release profile of the active substance. is there.

  Further, it is expected that the stability varies depending on the particle size of the fine particles. Therefore, it is possible to design a sustained release profile of the active substance by adjusting the particle size distribution of the fine particles containing the active substance.

  In the sustained release composition of the present invention, when the active substance release process from the above-mentioned 3) active substance-containing microparticles is mainly used, the active substance-containing microparticles are substantially contained in the sustained-release composition of the present invention. Preferably it does not diffuse.

Further, in the present invention, 4) the hydrogel disintegration process can be combined with the controlled release of the active substance from the sustained-release composition of the present invention. The hydrogel of the present invention can control the degradation rate in a living body (intravascular, intraperitoneal, subcutaneous, on the wound surface, etc.).
This is because the physical interaction force (hydrophobic interaction, electrostatic interaction, hydrogen bond, crystal structure, etc.) that forms the hydrogel is affected by the target component, causing the hydrogel structure to collapse. For example, when the hydrogel of the present invention in which the hydrogel is formed by cross-linking by hydrophobic interaction is placed in the target, the amphiphile (for example, phospholipid in the living body) in the target is highly hydrogel-forming. As a result of weakening the hydrophobic interaction between the hydrogel-forming polymers by adsorbing to the hydrophobic part of the molecule, the hydrogel is broken and the hydrogel is decomposed.
It is also possible to control the decomposition rate of the hydrogel of the present invention by a chemical reaction within the subject. This is because a hydrogel-forming polymer can be decomposed by a hydrolysis reaction, an oxidation reaction, or the like. For example, in the living body, the hydrolysis reaction and oxidation reaction are further accelerated by the enzyme, and the collapse of the hydrogel is promoted. In order to make it easy to receive a hydrolysis reaction, for example, a hydrogel-forming polymer containing an ester bond can be suitably used. Specifically, a hydrogel-forming polymer partially containing polyester such as polyglycolic acid or polylactic acid can be preferably used. Moreover, in order to make it easy to receive an oxidative decomposition reaction, for example, a hydrogel-forming polymer containing an ether bond can be preferably used. Specifically, a hydrogel-forming polymer partially containing a polyether such as polyethylene glycol can be suitably used.

  When the sol-gel transition temperature of the hydrogel of the present invention is lowered, the hydrogel has a decay rate in vivo, and when the sol-gel transition temperature is raised, the hydrogel decays in vivo faster. In addition, if the concentration of the hydrogel-forming polymer in the hydrogel is increased, the hydrogel will remain in the living body for a long time, and if the concentration of the hydrogel-forming polymer in the hydrogel is decreased, the hydrogel will remain in the living body. Hydrogel disappears faster.

  In the hydrogel of the present invention, when the sol-gel transition temperature of the hydrogel of the present invention is lowered, the storage elastic modulus (G ′) of the hydrogel at the living body temperature (37 ° C.) is increased. Further, when the concentration of the hydrogel-forming polymer in the hydrogel is increased, the storage elastic modulus (G ′) of the hydrogel at the living body temperature (37 ° C.) is increased. That is, in order to control the decay rate in the living body, G ′ at 37 ° C. may be controlled.

In the measurement measurement of G ′, the following measurement conditions can be preferably used.
<Dynamic / loss elastic modulus measurement conditions>
Measuring instrument (trade name): Stress-controlled rheometer AR500, manufactured by TA Instruments Co. Sample solution amount: about 0.8 g
Shape and dimensions of measurement cell: acrylic parallel disk (diameter 4.0 cm), gap 600 μm,
Measurement frequency: 1Hz
Applied stress: in the linear region.

  The relationship between the remaining period of the hydrogel of the present invention in the living body and G ′ differs depending on the site in the living body, so it cannot be generally stated. For example, the disintegration period in the abdominal cavity and the G ′ at an observation frequency of 1 Hz According to the knowledge of the present inventors, the relationship is as follows.

  That is, the preferable range of G ′ for disappearing within 3 days is 10 to 500 Pa, and the preferable range of G ′ for remaining within 14 days is 200 to 1500 Pa, G for remaining over 14 days. A preferable range of 'is 400 to 10,000 Pa.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is limited by the claims, and is not limited by the following examples.

Production Example 1
10 g of a polypropylene oxide-polyethylene oxide copolymer (propylene oxide / ethylene oxide average polymerization degree of about 60/180, manufactured by Asahi Denka Kogyo Co., Ltd .: Pluronic F-127) was dissolved in 30 ml of dry chloroform, and in the presence of phosphorus pentoxide, Hexamethylene diisocyanate (0.13 g) was added, and the reaction was allowed to proceed for 6 hours under reflux at the boiling point. After distilling off the solvent under reduced pressure, the residue was dissolved in distilled water, and ultrafiltration was performed using an ultrafiltration membrane (Amicon PM-30) with a molecular weight cut off of 30,000 to obtain a high molecular weight polymer and a low molecular weight polymer. Fractionated. The obtained aqueous solution was frozen to obtain F-127 high polymer and F-127 low polymer.

  The F-127 high polymer (hydrogel-forming polymer of the present invention, TGP-1) obtained as described above was dissolved in distilled water at a concentration of 8% by mass under ice cooling. When this aqueous solution was gently heated, the viscosity gradually increased from 21 ° C. and solidified at about 27 ° C. to form a hydrogel. When this hydrogel was cooled, it returned to an aqueous solution at 21 ° C. This change was repeatedly observed reversibly. On the other hand, the F-127 low polymer dissolved in distilled water at a concentration of 8% by mass below freezing did not gel at all even when heated to 60 ° C. or higher.

Production Example 2
160 mol of ethylene oxide was added by cationic polymerization to 1 mol of trimethylolpropane to obtain a polyethylene oxide triol having an average molecular weight of about 7000.

  After dissolving 100 g of the polyethylene oxide triol obtained above in 1000 ml of distilled water, 12 g of potassium permanganate was gradually added at room temperature, and the reaction was allowed to proceed for about 1 hour. After removing the solid matter by filtration, the product was extracted with chloroform, and the solvent (chloroform) was distilled off under reduced pressure to obtain 90 g of a polyethylene oxide tricarboxylate.

  10 g of the polyethylene oxide tricarboxylate obtained above and 10 g of polypropylene oxide diamino (propylene oxide average polymerization degree: about 65, manufactured by Jefferson Chemical Co., USA, trade name: Jeffamine D-4000, cloud point: about 9 ° C.) After dissolving in 1000 ml of carbon tetrachloride and adding 1.2 g of dicyclohexylcarbodiimide, the mixture was reacted for 6 hours under reflux at the boiling point. After cooling the reaction solution and removing solids by filtration, the solvent (carbon tetrachloride) is distilled off under reduced pressure, and the residue is vacuum-dried, and the hydrogel of the present invention in which a plurality of polypropylene oxides and polyethylene oxides are combined. A forming polymer (TGP-2) was obtained. This was dissolved in distilled water at a concentration of 5% by mass under ice-cooling, and its sol-gel transition temperature was measured and found to be about 16 ° C.

Production Example 3
96 g of N-isopropylacrylamide (manufactured by Eastman Kodak), 17 g of N-acryloxysuccinimide (manufactured by Kokusan Chemical Co., Ltd.) and 7 g of n-butyl methacrylate (manufactured by Kanto Chemical Co., Ltd.) are dissolved in 4000 ml of chloroform, and nitrogen is added. After the substitution, 1.5 g of N, N′-azobisisobutyronitrile was added and polymerized at 60 ° C. for 6 hours. After the reaction solution was concentrated, it was reprecipitated (reprecipitated) in diethyl ether. The solid was collected by filtration and then vacuum dried to obtain 78 g of poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate).

  To the poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) obtained above, an excess of isopropylamine was added to obtain poly (N-isopropylacrylamide-co-n-butyl methacrylate). Obtained. The cloud point of this aqueous solution of poly (N-isopropylacrylamide-co-n-butyl methacrylate) was 19 ° C.

  Chloroform 10 g of the above poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) and 5 g of both ends aminated polyethylene oxide (molecular weight 6,000, manufactured by Kawaken Fine Chemical Co., Ltd.) It was dissolved in 1000 ml and reacted at 50 ° C. for 3 hours. After cooling to room temperature, 1 g of isopropylamine was added and allowed to stand for 1 hour, and then the reaction solution was concentrated, and the residue was precipitated in diethyl ether. The solid matter was collected by filtration and then vacuum-dried to form the hydrogel-forming polymer (TGP-3) of the present invention in which a plurality of poly (N-isopropylacrylamide-co-n-butyl methacrylate) and polyethylene oxide were bonded. )

  The TGP-3 thus obtained was dissolved in distilled water at a concentration of 5% by mass under ice cooling, and the sol-gel transition temperature was measured to be about 21 ° C.

Production Example 4
(Sterilization method)
2.0 g of the above-described hydrogel-forming polymer (TGP-3) of the present invention is put into an EOG (ethylene oxide gas) sterilization bag (trade name: hybrid sterilization bag, manufactured by Hogi Medical Co., Ltd.), and an EOG sterilizer ( The bag was filled with EOG with Easy Pack (manufactured by Inoue Seieido) and left at room temperature all day and night. Further, after standing at 40 ° C. for half a day, the EOG was removed from the bag and aerated. The bag was sterilized by placing it in a vacuum dryer (40 ° C.) and leaving it for half a day with occasional aeration.

It was separately confirmed that the sol-gel transition temperature of the polymer was not changed by this sterilization operation.
Production Example 5
After dissolving 37 g of N-isopropylacrylamide, 3 g of n-butyl methacrylate and 28 g of polyethylene oxide monoacrylate (molecular weight 4,000, manufactured by NOF Corporation: PME-4000) in 340 ml of benzene, 2,2 ′ -0.8 g of azobisisobutyronitrile was added and reacted at 60 ° C for 6 hours. The obtained reaction product was dissolved by adding 600 ml of chloroform, and the solution was added dropwise to 20 L (liter) of ether to cause precipitation. The obtained precipitate was recovered by filtration, and the precipitate was vacuum-dried at about 40 ° C. for 24 hours, and then dissolved again in 6 L of distilled water, and a hollow fiber ultrafiltration membrane (H1P100 manufactured by Amicon Co., Ltd.) having a molecular weight cut-off of 100,000. -43) and concentrated to 2 liters at 10 ° C. The concentrated solution was diluted by adding 4 l of distilled water, and the above dilution operation was repeated. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and lyophilized to obtain 60 g of the hydrogel-forming polymer (TGP-4) of the present invention having a molecular weight of 100,000 or more. Obtained.

  1 g of the hydrogel-forming polymer (TGP-4) of the present invention obtained as described above was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 25 ° C.

Production Example 6
When the hydrogel-forming polymer (TGP-3) of Production Example 3 of the present invention was dissolved in distilled water at a concentration of 10% by mass and measured at η at 37 ° C., it was 5.8 × 10 ⁵ Pa · sec. there were. On the other hand, after agar was dissolved in distilled water at a concentration of 2% by mass at 90 ° C. and gelled at 10 ° C. for 1 hour, η at 37 ° C. was measured. 10 ⁷ Pa · sec).

Production Example 7
71.0 g of N-isopropylacrylamide and 4.4 g of n-butyl methacrylate were dissolved in 1117 g of ethanol. An aqueous solution obtained by dissolving 22.6 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) in 773 g of water was added thereto, and the mixture was heated to 70 ° C. under a nitrogen stream. While maintaining 70 ° C. in a nitrogen stream, 0.8 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 8 mL of 10% ammonium persulfate (APS) aqueous solution were added and stirred for 30 minutes. Further, 0.8 mL of TEMED and 8 mL of 10% APS aqueous solution were added four times at 30-minute intervals to complete the polymerization reaction. After cooling the reaction solution to 10 ° C. or lower, 5 L of 10 ° C. cold distilled water was added for dilution, and the mixture was concentrated to 2 L at 10 ° C. using an ultrafiltration membrane having a fractional molecular weight of 100,000.

  The concentrated solution was diluted by adding 4 L of cold distilled water, and the above ultrafiltration concentration operation was performed again. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and lyophilized to obtain 72 g of the hydrogel-forming polymer (TGP-5) of the present invention having a molecular weight of 100,000 or more. Obtained.

  1 g of the hydrogel-forming polymer (TGP-5) of the present invention obtained as described above was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 20 ° C.

Production Example 8
42.0 g of N-isopropylacrylamide and 4.0 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution in which 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) was dissolved in 65.1 g of water was added thereto, and the mixture was heated to 70 ° C. under a nitrogen stream. While maintaining 70 ° C. in a nitrogen stream, 0.4 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 4 mL of 10% ammonium persulfate (APS) aqueous solution were added and stirred for 30 minutes. Furthermore, 0.4 mL of TEMED and 4 mL of 10% APS aqueous solution were added four times at 30 minute intervals to complete the polymerization reaction. The reaction solution was cooled to 5 ° C. or lower, diluted by adding 5 L of 5 ° C. cold distilled water, and concentrated to 2 L at 5 ° C. using an ultrafiltration membrane having a fractional molecular weight of 100,000.

  The concentrated solution was diluted by adding 4 L of cold distilled water, and the above ultrafiltration concentration operation was performed again. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and lyophilized to obtain 40 g of the hydrogel-forming polymer (TGP-6) of the present invention having a molecular weight of 100,000 or more. Obtained.

  1 g of the hydrogel-forming polymer (TGP-6) of the present invention obtained as described above was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 7 ° C.

Production Example 9
45.5 g of N-isopropylacrylamide and 0.56 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution in which 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) was dissolved in 65.1 g of water was added thereto, and the mixture was heated to 70 ° C. under a nitrogen stream. While maintaining 70 ° C. in a nitrogen stream, 0.4 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 4 mL of 10% ammonium persulfate (APS) aqueous solution were added and stirred for 30 minutes. Furthermore, 0.4 mL of TEMED and 4 mL of 10% APS aqueous solution were added four times at 30 minute intervals to complete the polymerization reaction. After cooling the reaction solution to 10 ° C. or lower, 5 L of 10 ° C. cold distilled water was added for dilution, and the mixture was concentrated to 2 L at 10 ° C. using an ultrafiltration membrane having a fractional molecular weight of 100,000.

  The concentrated solution was diluted by adding 4 L of cold distilled water, and the above ultrafiltration concentration operation was performed again. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and lyophilized to obtain 22 g of the hydrogel-forming polymer (TGP-7) of the present invention having a molecular weight of 100,000 or more. Obtained.

  1 g of the hydrogel-forming polymer (TGP-7) of the present invention obtained as described above was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 37 ° C.

Example 1
The hydrogel-forming polymers TGP-5, TGP-6, and TGP-7 obtained in Production Examples 7, 8, and 9 were each dissolved in physiological saline to prepare a 10 wt% solution. Each sol-gel transition temperature was measured and found to be 18 ° C. (TGP-5), 5 ° C. (TGP-6), and 35 ° C. (TGP-7). Each physiological saline solution was cooled below its sol-gel transition temperature, and 20 mL / kg was administered intraperitoneally to 10 groups of 10 6-week-old rats (5 males and 5 females).

  The administration method was performed by using a syringe and an indwelling needle (22G) after removing the hair of the rat abdomen with an electric clipper and disinfecting the administration site with ethanol for disinfection. All groups showed a body weight increase equivalent to the group administered with 20 mL / kg of physiological saline intraperitoneally as a control, and no abnormal findings were observed during the 7-day observation period. Seven days after administration, the blood was lethal under ether anesthesia, and all organs and tissues were examined for the presence of abnormalities and the remaining hydrogel (the regenerating material of the present invention) in the abdominal cavity. None of the groups showed any abnormalities in all organs and tissues. For TGP-5 and TGP-6, residual hydrogel was observed in the abdominal cavity. However, for TGP-7 having a high sol-gel transition temperature of 35 ° C., the remaining hydrogel remained in the abdominal cavity on the seventh day after administration. I did not admit.

Example 2
Each of the hydrogel-forming polymers TGP-5 and TGP-6 obtained in Production Examples 7 and 8 was dissolved in physiological saline to prepare a 10 wt% solution. Each physiological saline solution was cooled below its sol-gel transition temperature, and 1 mL was injected subcutaneously into the back of the rat. The administration method was performed by using a syringe and an indwelling needle (22G) after removing the hair of the back of the rat with an electric clipper and disinfecting the administration site with ethanol for disinfection.

  TGP-5 having a sol-gel transition temperature of 18 ° C. disappeared from the subcutaneous rat on the 23rd day after administration, and its residual was not observed macroscopically. On the other hand, when TGP-6 having a sol-gel transition temperature of 5 ° C. was necropsied on the 37th day after administration, the residual was visually observed, and no abnormal findings such as foreign body reaction were observed in the surrounding tissue even in the HE-stained tissue image. It was.

Example 3
The hydrogel-forming polymer TGP-5 obtained in Production Example 7 was dissolved in physiological saline, and the polymer concentration was 10 mass% (wt%), 8 mass% (wt%), 6 mass% (wt%). A solution of was prepared. Each sol-gel transition temperature was measured and found to be 18 ° C., 20 ° C., and 22 ° C., respectively. Each physiological saline solution was cooled below its sol-gel transition temperature, and 1 mL / kg was administered intraperitoneally to 10 groups of 10 6-week-old rats (5 males and 5 females). The administration method was performed by using a syringe and an indwelling needle (22G) after removing the hair of the rat abdomen with an electric clipper and disinfecting the administration site with ethanol for disinfection. On the 1st day, 3rd day, 7th day, 14th day and 21st day after the administration, 2 mice (1 male, 1 female) in each group were exsanguinated under ether anesthesia. Residuals were inspected. A hydrogel having a polymer concentration of 6% by mass (wt%) disappeared from the abdominal cavity on the third day after administration, and a hydrogel having a polymer concentration of 8% by mass (wt%) disappeared from the abdominal cavity on the 14th day after administration. The hydrogel having a polymer concentration of 10% by mass (wt%) disappeared from the abdominal cavity on the 21st day after administration.

Example 4
1 g of TGP-5 was dissolved in 4 g of 5% glucose injection under ice cooling to prepare a 20 wt% TGP-5 aqueous solution. 2 g of this 20 wt% TGP-5 aqueous solution and 2 g of a liposome preparation (Doxil (registered trademark), DOX concentration: 2 mg / ml, Ortho Biotech Products LP, USA) encapsulating doxorubicin hydrochloride (DOX). To obtain a sustained-release composition I of the present invention containing fine particles (liposomes) containing DOX which is an anticancer agent as an active substance (TGP-5 concentration 10 wt%, DOX concentration 1 mg / ml). A 6-well cell culture insert (Transwell (registered trademark) 3428, Cornig Inc. USA) having 0.8 g of the sustained-release composition I of the present invention in a liquid state under ice cooling and having a polycarbonate membrane (diameter 24 mm) having a pore size of 8 μm as the bottom surface. ) And heated to 37 ° C. to obtain a disc-shaped hydrogel having a diameter of 24 mm (area 4.52 cm ² ) and a thickness of 1.77 mm. 3 ml of physiological saline was placed in 1 well of a 6-well cell culture plate, and the above-mentioned disc-shaped hydrogel was placed on the bottom at 37 ° C., and the cell culture insert was inserted.

  While shaking at 37 ° C., a total amount of 3 ml of physiological saline in the well was collected every predetermined time, and 37 ° C. physiological saline was newly added. The DOX concentration in the collected physiological saline was determined by absorbance measurement at 480 nm, and the cumulative elution rate (Mt / M∞) up to a predetermined time is shown in Tables 1-3. The experiment was performed three times, and the average value and standard deviation were shown together. The results of Tables 1 to 3 are shown in the graphs of FIGS.

As shown in FIG. 1, it can be seen that the sustained release of the active substance from the sustained release composition of the present invention is sustainedly released at a substantially constant rate from the 5th day to the 27th day. This indicates that an ideal zero-order release (drug release rate is constant) as the sustained release characteristics of the drug. Further, the square root and the plot of the horizontal axis elapsed time (in seconds) (Fig. 2), not to Mt / M∞ = (Dt / π ) 1/2 × 4 / L (1) expression of the proportional relationship, the It is clear that the sustained release of the active substance from the sustained release composition of the invention is not a release due to a simple diffusion phenomenon in the hydrogel of DOX. It was confirmed that about 50% of the DOX content was released in 27 days, and sustained release of the active substance continued for a very long time.

Comparative Example 1
A freeze-dried preparation of doxorubicin hydrochloride (containing 10 mg of DOX) was dissolved in 10 ml of 5% glucose injection solution at room temperature, and 9 g thereof was added to 1 g of TGP-5 and dissolved under ice cooling (TGP-5 concentration: 10 wt%, DOX). Concentration 1 mg / ml). 0.844 g of this solution was poured into a 6-well cell culture insert (Transwell (registered trademark) 3428, Cornig Inc. USA) having a bottom surface of a polycarbonate membrane (diameter 24 mm) having a pore size of 8 μm in a liquid state under ice-cooling. To a disc-shaped hydrogel having a diameter of 24 mm (area: 4.52 cm ² ) and a thickness of 1.87 mm. 3 ml of physiological saline was placed in 1 well of a 6-well cell culture plate, and the above-mentioned disc-shaped hydrogel was placed on the bottom at 37 ° C., and the cell culture insert was inserted. While shaking at 37 ° C., a total amount of 3 ml of physiological saline in the well was collected every predetermined time, and 37 ° C. physiological saline was newly added. The DOX concentration in the collected physiological saline was determined by absorbance measurement at 480 nm, and the cumulative elution rate (Mt / M∞) up to a predetermined time is shown in Tables 4 to 5 and FIG. The elution rate exceeded 90% in 12 hours.

From the results of Tables 4 to 5, when the elution time t1 ^{/ 2} and the cumulative elution rate (Mt / M∞) are plotted in the range of the cumulative elution rate up to 60% and approximated by the least square method as a linear function of y = ax, straight line shown in FIG. 4 (y = 0.006x) is obtained, the correlation coefficient ^{R 2} was 0.9939. That is, the relationship of Mt / M∞ = (Dt / π) ^1/2 × 4 / L (1) is established, and the elution of DOX depends only on the diffusion phenomenon of DOX molecules in the gel. Is clear. Here, in consideration of elution from only one side of the gel, when L = 0.187 × 2 = 0.373 cm and the diffusion coefficient D of the DOX molecule in the gel is determined, D = 9.8 × 10 ⁻⁷ ( cm ² / sec).

Example 5
1 g of TGP-5 was dissolved in 4 g of 5% glucose injection under ice cooling to prepare a 20 wt% TGP-5 aqueous solution. 2 g of this 20 wt% TGP-5 aqueous solution and 2 g of a liposome preparation (Doxil (registered trademark), DOX concentration: 2 mg / ml, Ortho Biotech Products L. P., USA) encapsulating doxorubicin HCl (DOX). Mixing under ice-cooling, a sustained release composition I of the present invention containing fine particles (liposomes) containing DOX as an anticancer agent as an active substance was obtained (TGP-5 concentration 10 wt%, DOX concentration 1 mg / ml). 0.1 g of the sustained release composition I of the present invention was injected subcutaneously into nude mice in a liquid state under ice cooling. As a result of necropsy on the 25th day, it was confirmed that the sustained-release composition of the present invention remained in an orange state, and DOX remained in the sustained-release composition of the present invention.

Comparative Example 2
A freeze-dried preparation of doxorubicin hydrochloride (containing 10 mg of DOX) was dissolved in 10 ml of 5% glucose injection solution at room temperature, and 9 g thereof was added to 1 g of TGP-5 and dissolved under ice cooling (TGP-5 concentration: 10 wt%, DOX). Concentration 1 mg / ml). 0.1 g of this solution was injected subcutaneously into nude mice in a liquid state under ice cooling. An autopsy on the 7th day confirmed that the hydrogel remained in a completely transparent state, and that all the DOX had been released within 7 days.
Reference example 1
A liposome preparation (Doxil (registered trademark), DOX concentration: 2 mg / ml, Ortho Biotech Products L. P., USA) encapsulating doxorubicin hydrochloride (doxorubicin HCL, DOX) was diluted 30-fold with 5% glucose injection solution. When a visible light absorption spectrum was measured, the maximum absorption wavelength was 496 nm, and the absorbance was 0.948. When 0.1 mL of 10 wt% Triton X100 aqueous solution was added to 3 mL of this solution to break the liposome and the visible light absorption spectrum was measured again, the maximum absorption wavelength was 480 nm and the absorbance was 1.301. That is, there is a difference in the visible light absorption spectrum between the free state and the state encapsulated in the liposome, and it can be determined whether the DOX is encapsulated in the liposome or eluted from the liposome by measuring the visible light absorption spectrum.
Reference example 2
A liposomal preparation (Doxil (registered trademark), DOX concentration: 2 mg / ml, Ortho Biotech Products L.P., USA) 0.8 mL encapsulating doxorubicin hydrochloride (doxorubicin HCL, DOX). 0.1 mL of the filtrate filtered through a membrane (Millipore) was collected, diluted 30-fold with 5% glucose injection, and the visible light absorption spectrum was measured. The maximum absorption wavelength was 480 nm, and the absorbance was 0.012. Met. That is, it was confirmed that DOX existing outside the liposome in the above-mentioned liposome preparation Doxil was 0.9%, and 99% or more was encapsulated in the liposome.
Next, the liposome preparation Doxil was allowed to stand at 37 ° C. for 22 days, and then 0.1 mL of a filtrate obtained by filtering 0.8 mL of the liposome preparation Doxil through an ultrafiltration membrane (Millipore) with a molecular weight cut off of 10,000 was collected. When the visible light absorption spectrum was measured after 30-fold dilution with 5% glucose injection, the maximum absorption wavelength was 480 nm and the absorbance was 0.017. Accordingly, the DOX eluted from the liposomes after standing at 37 ° C. for 22 days is only 0.4% of the DOX encapsulated in the liposomes.
On the other hand, surprisingly, the sustained-release composition of the present invention shown in Example 4 was able to release about 44% of DOX after standing at 37 ° C. for 22 days. This surprising effect is considered to be due to the presence of the interaction between the hydrogel-forming polymer and the fine particles containing the active substance in the sustained-release composition of the present invention.

It is a graph which shows the DOX sustained release behavior from the hydrogel containing the DOX containing liposome of this invention (a horizontal axis is elapsed days). It is a graph which shows the DOX sustained release behavior from the hydrogel containing the DOX containing liposome of this invention (a horizontal axis is the square root of elapsed seconds). It is a graph which shows DOX sustained release from a DOX containing hydrogel (a horizontal axis is elapsed time (hour)). It is a graph which shows DOX sustained release from a DOX containing hydrogel (a horizontal axis is a square root of elapsed seconds).

Claims (10)

A hydrogel-forming polymer capable of forming a hydrogel having a sol-gel transition temperature;
With a dispersion liquid;
Including at least fine particles containing an active substance;
A sustained-release composition characterized by being in a fluid sol state at a temperature lower than the sol-gel transition temperature and reversibly in a hydrogel state at a temperature higher than the sol-gel transition temperature.   The sustained-release composition according to claim 1, wherein the microparticles themselves are microparticles capable of expressing a sustained-release property of an active substance.   The sustained release composition according to claim 1 or 2, wherein the sol-gel transition temperature is in the range of 0 ° C to 37 ° C.   The sustained-release composition according to any one of claims 1 to 3, wherein the active substance is a physiologically active substance.   The sustained-release composition according to any one of claims 1 to 4, which is substantially water-insoluble in a hydrogel state.   The sustained-release composition according to any one of claims 1 to 5, wherein the fine particles have a particle size of 1 nm or more.   The sustained release composition according to any one of claims 1 to 6, wherein the fine particles are selected from the group consisting of liposomes, dendrimers, fat preparations, microcapsules, microspheres, and polymeric micelles.   The sustained-release composition according to claim 7, wherein the fine particles are liposomes.   The sustained-release composition according to claim 4, wherein the physiologically active substance is selected from the group consisting of anticancer agents, antibiotics, growth factors, immunopotentiators, immunosuppressants, and antithrombotic agents. A hydrogel-forming polymer capable of forming a hydrogel having a sol-gel transition temperature; at least a dispersion liquid; and fine particles containing an active substance; a fluid sol state at a temperature lower than the sol-gel transition temperature And a sustained release composition that reversibly becomes a hydrogel state at a temperature higher than the sol-gel transition temperature,
Placing the sustained release composition at a temperature lower than the fluid sol-gel transition temperature at an application site where sustained release should be developed;
A sustained release method characterized in that the active substance is sustainedly released from the sustained release composition by gelation at a temperature higher than the sol-gel transition temperature.

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