JP7598604B2 - Treatment agent for urethral stricture and treatment method for urethral stricture - Google Patents

Description

The present invention relates to a therapeutic agent useful for treating urethral stricture. The present invention also relates to a method for treating urethral stricture.

Urethral stricture can occur for a variety of reasons, including aftereffects of endoscopic urethral surgery for benign prostatic hyperplasia or bladder cancer, trauma from traffic or workplace accidents, and hypospadias, a congenital urethral disease. In this condition, the urethral mucosa is damaged by injury or inflammation, and in the process of repairing the damage, scarring occurs in the urethral mucosa and the corpus spongiosum that surrounds the urethral mucosa, causing the urethra to narrow.

There is a method of treating urethral stricture that involves surgical reconstruction of the urethra, but this is highly invasive and requires long-term hospitalization, so in recent years, transurethral endoscopic dilation techniques have been performed that are less invasive and simpler to use, such as bougies (urethral dilators), balloon catheters, cold knives, and lasers (Hayashida Shigeaki, Kiriyama Suguo, Hironaka Hiroshi, Yoshikawa Shizuo, Experience of internal urethral incision for male urethral stricture, Annals of Urology (1972), 18 (8): 588-593).

Shigeaki Hayashida, Suguo Kiriyama, Hiroshi Hironaka, Shizuo Yoshikawa, Experience of Internal Urethral Incision for Male Urethral Stricture, Annals of Urology (1972), 18(8): 588-593

However, the above conventional treatment methods have an extremely low ability to reconstruct epithelial cells on the scar-tissued inner surface of the urethra, resulting in the problem of recurrence of urethral stricture. The present invention aims to provide a urethral stricture treatment agent and a urethral stricture treatment method that can avoid restenosis using a minimally invasive transurethral endoscopic technique in the treatment of urethral stricture.

The inventors have discovered that placing a hydrogel with specific physical properties on the inner surface of the urethra after incision treatment using a transurethral endoscopic technique as a treatment for urethral stricture promotes epithelialization of the incision site and effectively prevents recurrence of urethral stricture due to scar tissue formation at the site, and thus completed the present invention.

Furthermore, the inventors have found that incorporating animal cells into the hydrogel is effective in solving the above problems. They have found that it is particularly effective if the animal cells are the patient's own oral mucosa cells.

In other words, the problem of the present invention is solved by a urethral stricture treatment agent that contains at least a hydrogel-forming polymer and is characterized by having a storage modulus of 50 Pa or less at 10°C and a storage modulus of 100 Pa or more at 37°C.

The object of the present invention is further achieved by a urethral stricture treatment agent, which is characterized in that the urethral stricture treatment agent contains animal cells.

The object of the present invention is also achieved by a urethral stricture treatment agent, characterized in that the animal cells are the patient's own oral mucosal cells.

Furthermore, the object of the present invention is achieved by a method for treating urethral stricture, which comprises at least the steps of injecting a urethral stricture treatment agent, which contains at least a hydrogel-forming polymer and has a storage modulus of 50 Pa or less at 10°C and 100 Pa or more at 37°C, cooled to a temperature of 10°C or less, into the inner surface of the urethra that has been incised by a transurethral endoscopic technique, and then retaining the agent on the inner surface of the urethra at a temperature of room temperature or higher.

As described above, according to the present invention, by placing a hydrogel with specific physical properties on the inner surface of the urethra that has been incised using a transurethral endoscopic technique, epithelialization of the incision site can be promoted, and the recurrence of urethral stricture due to scar tissue formation at that site can be effectively prevented.

Furthermore, the hydrogel contains animal cells (especially the patient's own oral mucosal cells), which further promotes epithelialization of the incision site on the inner surface of the urethra.

The present invention will be described in detail below.
(Hydrogel-forming polymers)
The "hydrogel-forming polymer" of the present invention refers to a polymer that has the property of thermoreversibly forming a crosslinking structure or a network structure and, based on the structure, thermoreversibly forming a hydrogel that retains a dispersed liquid such as water inside. Also, the term "hydrogel" refers to a gel that contains a crosslinking or network structure made of a polymer and water supported or retained in the structure.

(Storage Modulus)
In the present invention, the storage modulus of the hydrogel can be measured by the method described in the literature (H. Yoshioka et al., Journal of Macromolecular Science, A31(1), 113 (1994)). That is, the dynamic modulus of a sample at an observation frequency of 1 Hz is measured at a specified temperature (10°C, 25°C or 37°C) to determine the storage modulus (G', elastic term) of the sample. The following measurement conditions can be suitably used for this measurement.

<Measurement conditions for dynamic and loss modulus>
Measurement equipment (product name): Stress-controlled rheometer AR500, manufactured by TA Instruments Amount of sample solution: Approximately 0.8 g
Shape and dimensions of the measurement cell: Acrylic parallel disk (diameter 4.0 cm), gap 600 μm
Measurement frequency: 1Hz
Applied stress: In the linear region.

The urethral stricture treatment agent of the present invention has a storage modulus of 50 Pa or less, preferably 30 Pa or less (particularly 10 Pa or less) at 10°C, and a storage modulus of 100 Pa or more, preferably 200 Pa or more (particularly 300 Pa or more) at 37°C.

The urethral stricture treatment agent of the present invention is injected into the urethral stricture treatment site at a low temperature of 10°C or less, and the urethral stricture treatment agent is left in place at the urethral stricture treatment site at body temperature. If the storage modulus of the urethral stricture treatment agent at 10°C exceeds 50 Pa, the agent becomes too hard and difficult to inject through a catheter.

On the other hand, if the storage modulus of the urethral stricture treatment agent of the present invention at 37°C is less than 100 Pa, its strength will be insufficient, making it difficult to leave it in the urethral stricture treatment site for a long period of time.

Furthermore, in the case of male patients, the site of urethral stricture treatment is often located in the penis, which is exposed to the outside of the body and is therefore easily affected by the outside temperature. If the storage modulus of the aqueous solution of the "hydrogel-forming polymer" of the present invention at room temperature (25°C) is below 100 Pa, it will easily become fluid due to a drop in the outside temperature, resulting in incomplete placement of animal cells at the site of urethral stricture treatment. As a result, the ability to reconstruct epithelial cells on the scarred inner surface of the urethra is low, which causes the problem of recurrence of urethral stricture. Therefore, it is preferable that the storage modulus of the urethral stricture treatment agent of the present invention is 100 Pa or more at 25°C, preferably 200 Pa or more (particularly 300 Pa or more).

The "hydrogel-forming polymer" that imparts the above-mentioned suitable storage modulus to the urethral stricture treatment agent of the present invention can be easily selected from the specific compounds described below according to the above-mentioned screening method (storage modulus measurement method).

Specific examples of polymers whose hydrogels exhibit reversible fluidity at lower temperatures include polyalkylene oxide block copolymers, such as block copolymers of polypropylene oxide and polyethylene oxide; etherified celluloses, such as methyl cellulose and hydroxypropyl cellulose; and chitosan derivatives (K. R. Holme, et al. Macromolecules, 24, 3828 (1991)).

(Suitable hydrogel-forming polymers)
A hydrogel-forming polymer that utilizes hydrophobic bonds for crosslinking and that can be suitably used as the "hydrogel-forming polymer" of the present invention is preferably formed by bonding multiple blocks having a cloud point to a hydrophilic block.

The hydrophilic blocks are preferably present so that the hydrogel becomes water soluble at lower temperatures, and the cloud point blocks are preferably present so that the hydrogel changes to a gel state at higher temperatures.
In other words, a block having a cloud point dissolves in water at a temperature lower than the cloud point and becomes insoluble in water at a temperature higher than the cloud point, and therefore, at a temperature higher than the cloud point, the block serves as a crosslinking point consisting of a hydrophobic bond to form a gel.

The hydrogel used in the present invention utilizes the property that hydrophobic bonds not only become stronger with increasing temperature, but that this change is reversible with respect to temperature. In order to form multiple crosslinking points within one molecule and form a gel with excellent stability, it is preferable that the "hydrogel-forming polymer" has multiple "blocks with a cloud point."

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

Furthermore, it is preferable that the "hydrogel-forming polymer" used in the present invention is one that is decomposed and absorbed in the body. In other words, it is preferable that the "hydrogel-forming polymer" of the present invention is decomposed in the body by a hydrolysis reaction or an enzymatic reaction, and is absorbed and excreted as a low molecular weight substance that is harmless to the body.

When the "hydrogel-forming polymer" of the present invention is composed of multiple blocks having a cloud point and a hydrophilic block bonded together, it is preferable that at least one of the blocks having a cloud point and the hydrophilic block, and preferably both, are degraded and absorbed in the body.

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

In order to make a block having a cloud point degradable and absorbable in the body, it is effective to make the block having a cloud point a polypeptide consisting of hydrophobic and hydrophilic amino acids. Alternatively, polyester-type biodegradable polymers such as polylactic acid and polyglycolic acid can be used as a block having a cloud point that is degradable and absorbed in the body.

It is preferable that the cloud point of the above polymer (block having a cloud point) is higher than 4°C and lower than 40°C in order to obtain a desired storage modulus at a specified temperature for the polymer used in the present invention (a compound in which multiple blocks having a cloud point and a hydrophilic block are combined).

The cloud point can be measured, for example, by cooling an aqueous solution of about 1% by mass of the above polymer (block having a cloud point) to form a transparent homogeneous solution, and then gradually increasing the temperature (at a rate of about 1°C/min) until the point at which the solution first becomes cloudy is determined 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-acryloylpiperidine; poly-N-n-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 above polymer may be a homopolymer or a copolymer of the monomer constituting the above polymer with another monomer. As the other monomer constituting such a copolymer, either a hydrophilic monomer or a hydrophobic monomer can be used. In general, when copolymerized with a hydrophilic monomer, the cloud point of the product increases, and when copolymerized with a hydrophobic monomer, the cloud point of the product decreases. Therefore, by selecting the monomer to be copolymerized, a polymer having a desired cloud point (for example, a cloud point higher than 4° C. and lower than 40° C.) can be obtained.

(hydrophilic monomer)
Examples of the hydrophilic monomer include, but are not limited to, N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methylacrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, acrylic acid, methacrylic acid and salts thereof having an acidic group, vinyl sulfonic acid, styrene sulfonic acid, etc., as well as N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methacrylate, N,N-dimethylaminopropyl acrylamide and salts thereof having a basic group.

(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 alkyl methacrylamide derivatives such as N-n-butyl methacrylamide, vinyl chloride, acrylonitrile, styrene, and vinyl acetate, but are not limited to these.

(hydrophilic block)
On the other hand, specific examples of the hydrophilic block to be bonded to the block having the above-mentioned cloud point include methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinylpyrrolidone, polyvinylpyridine, polyacrylamide, polymethacrylamide, poly N-methylacrylamide, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid, and salts thereof; poly N,N-dimethylaminoethyl methacrylate, poly N,N-diethylaminoethyl methacrylate, poly N,N-dimethylaminopropylacrylamide, and salts thereof.

Furthermore, it is desirable for the hydrophilic block to be decomposed, metabolized, and excreted in the body, 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 of bonding the block having a cloud point and the above-mentioned hydrophilic block is not particularly limited, but for example, it can be carried out by introducing a polymerizable functional group (e.g., acryloyl group) into one of the above-mentioned blocks and copolymerizing a monomer that gives the other block. In addition, the bond between the block having a cloud point and the above-mentioned hydrophilic block can also be obtained by block copolymerization of a monomer that gives the block having a cloud point and a monomer that gives the hydrophilic block. In addition, the bond between the block having a cloud point and the hydrophilic block can also be carried out by introducing reactive functional groups (e.g., hydroxyl group, amino group, carboxyl group, isocyanate group, etc.) into both in advance and bonding them by chemical reaction. In this case, multiple reactive functional groups are usually introduced into the hydrophilic block. In addition, the bond between polypropylene oxide having a cloud point and a hydrophilic block can be obtained, for example, by repeatedly sequentially polymerizing propylene oxide and a monomer (e.g., ethylene oxide) constituting the "other hydrophilic block" by anionic polymerization or cationic polymerization, thereby obtaining a block copolymer in which polypropylene oxide and a "hydrophilic block" (e.g., polyethylene oxide) are bonded. Such a block copolymer can also be obtained by introducing a polymerizable group (e.g., acryloyl group) into the end of polypropylene oxide and then copolymerizing a monomer constituting the hydrophilic block. Furthermore, the polymer used in the present invention can also be obtained by introducing a functional group capable of bonding and reacting with the functional group (e.g., hydroxyl group) at the end of polypropylene oxide into the hydrophilic block and reacting the two. In addition, the "hydrogel-forming polymer" used in the present invention can also be obtained by linking a material 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.

In the polymer of the present invention in an embodiment that includes a block having a cloud point, the "block having a cloud point" present in the molecule is water-soluble together with the hydrophilic block at a temperature lower than the cloud point, and therefore completely dissolves in water and exhibits a sol state. However, when the temperature of an aqueous solution of this polymer is heated to a temperature higher than the cloud point, the "block having a cloud point" present in the molecule becomes hydrophobic, and separate molecules associate with each other through hydrophobic interactions.

On the other hand, since the hydrophilic blocks are water-soluble even at this time (when heated to a temperature higher than the cloud point), the polymer of the present invention forms a hydrogel in water having a three-dimensional network structure in which the hydrophobic associations between the blocks having cloud points serve as crosslinking points. When the temperature of this hydrogel is again cooled to a temperature lower than the cloud point of the "block having a cloud point" present in the molecule, the block having the cloud point becomes water-soluble, the crosslinking points due to the hydrophobic associations are released, the hydrogel structure disappears, and the "hydrogel-forming polymer" of the present invention becomes a complete aqueous solution again. Thus, the change in physical properties of the polymer of the present invention in a preferred embodiment is based on the reversible change in hydrophilicity and hydrophobicity of the block having a cloud point present in the molecule at the cloud point, and is therefore completely reversible in response to temperature changes.

According to the inventors' research, it is believed that the delicate balance of hydrophilicity and hydrophobicity of the above-mentioned "hydrogel-forming polymer" in water contributes to the stability of cells when they are cultured.

(Solubility of gel)
As described above, the hydrogel-forming polymer of the present invention is substantially water-insoluble at body temperature (37° C.) and reversibly water-soluble under ice-cooling. The term "substantially water-insoluble" means that the amount of the polymer dissolved in 100 mL of water at 37° C. is preferably 5.0 g or less (more preferably 0.5 g or less, and particularly preferably 0.1 g or less).

On the other hand, the above-mentioned "water-soluble" under ice-cooling means that the amount of the polymer that dissolves in 100 mL of water at 10° C. is preferably 0.5 g or more (even more preferably 1.0 g or more).
Furthermore, "reversibly exhibiting water solubility" means that even after an aqueous solution of the "hydrogel-forming polymer" once becomes a "substantially water-insoluble" gel state at 37°C, the aqueous solution still exhibits the above-mentioned water solubility at 10°C.

The above polymer preferably exhibits a viscosity of 10 to 3,000 centipoise (more preferably 50 to 1,000 centipoise) in a 10% aqueous solution at 5° C. Such a viscosity is preferably measured, for example, under the following measurement conditions.
Viscometer: Stress-controlled rheometer (model: AR500, manufactured by TA Instruments)
Rotor diameter: 60mm
Rotor shape: parallel plate

The aqueous solution of the "hydrogel-forming polymer" of the present invention does not substantially dissolve even when immersed in a large amount of water at 37°C. The above characteristics of the hydrogel formed by the "hydrogel-forming polymer" can be confirmed, for example, as follows. That is, 0.15 g of the "hydrogel-forming polymer" is dissolved in 1.35 g of distilled water under ice cooling to prepare a 10 wt% aqueous solution, and the aqueous solution is poured into a plastic petri dish with a diameter of 35 mm and humidified to 37°C to form a gel with a thickness of about 1.5 mm in the petri dish, and the weight (f grams) of the entire petri dish containing the gel is then measured. Next, the entire petri dish containing the gel is left to stand in 250 ml of water at 37°C for 10 hours, and the weight (g grams) of the entire petri dish containing the gel is measured to evaluate whether or not the gel has dissolved from the gel surface. In this case, in the hydrogel-forming polymer of the present invention, the weight loss rate of the gel, i.e., (f-g)/f, is preferably 5.0% or less, and more preferably 1.0% or less (particularly 0.1% or less).

After gelling at 37°C, an aqueous solution of the "hydrogel-forming polymer" of the present invention will not dissolve even if it is immersed in a large amount of water (about 0.1 to 100 times the volume of the gel) for a long period of time. Such properties of the polymer used in the present invention are achieved, for example, by the presence of two or more (multiple) blocks with cloud points within the polymer.

In contrast, the inventors have found that when a similar gel is made using the aforementioned Pluronic F-127, which is made of polypropylene oxide bonded to both ends, the gel dissolves completely in water after being left to stand for several hours.

In order to keep cytotoxicity in the non-gelled state as low as possible, it is preferable to use a "hydrogel-forming polymer" that can gel at a concentration relative to water of 20% or less (preferably 15% or less, and especially 10% or less), i.e., {(polymer)/(polymer+water)} x 100 (%).

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

The storage modulus of the urethral stricture treatment agent of the present invention can be adjusted to a preferred range by adjusting the concentration of the "hydrogel-forming polymer" in the urethral stricture treatment agent in addition to selecting the type of "hydrogel-forming polymer" as described above. Usually, the storage modulus of the urethral stricture treatment agent of the present invention increases when the concentration of the "hydrogel-forming polymer" is increased, and decreases when the concentration is decreased.

(Added salt)
The urethral stricture therapeutic agent of the present invention contains at least the above-mentioned "hydrogel-forming polymer", and it is desirable to add salts such as a pH buffer solution or physiological saline in order to give the agent a pH and osmotic pressure close to those of body fluids of the living body.

(Animal cells)
The urethral stricture treatment agent of the present invention can be used by dispersing animal cells. As the animal cells, urethral mucosal epithelial cells are most preferable. In order to facilitate collection and to avoid immune rejection, autologous oral mucosal cells are preferably used.

In addition to differentiated cells, undifferentiated cells can also be used because of their high immune tolerance as animal cells. Examples of undifferentiated cells include pluripotent stem cells such as ES cells and iPS cells, and mesenchymal stem cells.

The urethral stricture treatment agent of the present invention can be mixed with animal cells immediately before use as a urethral stricture treatment agent, but the cells can also be preliminarily dispersed in the urethral stricture treatment agent and allowed to grow before application to the site of urethral stricture.

If the above cells are undifferentiated cells, they can be proliferated in an undifferentiated state and then induced to differentiate into a lineage of mucosal epithelial cells.

(Cytokines)
The urethral stricture treatment agent of the present invention may contain various cytokines for the purpose of promoting mucosal cell epithelialization at the urethral stricture site. Since cytokines are water-soluble, they will easily diffuse if directly injected into the urethral stricture site. However, the urethral stricture treatment agent of the present invention forms a fine polymer network with the "hydrogel-forming polymer" and suppresses the diffusion of cytokines. Therefore, the cytokines can be retained at a high concentration around the urethral stricture site, and the effect of the cytokines can be maintained for a long period of time.

Cytokines suitable for use in the present invention can be used without particular limitations as long as they promote epithelialization of mucosal cells at the site of urethral stricture, but it is preferable to use those that have the effect of maintaining the undifferentiated state of cells, promoting proliferation, and promoting the induction of differentiation into the urethral mucosal epithelial cell lineage.

(urethral stricture treatment method)
The urethral stricture treatment agent of the present invention is placed on the inner surface of the urethra that has been incised by a transurethral endoscopic technique to promote epithelialization of the incision site, and the recurrence of urethral stricture due to scar tissue formation at the site. Effectively prevent.

A patient undergoes a standard transurethral endoscopic incision to incise the site of urethral stricture, and after inserting a urethral catheter, the urethral stricture treatment agent of the present invention, which has been cooled on ice, is injected between the area around the urethral catheter and the incision site on the inner surface of the urethra via a catheter separate from the urethral catheter. The urethral stricture treatment agent of the present invention is warmed by body heat and immediately gels, and is placed so as to cover the entire incision site on the inner surface of the urethra. When the urethral catheter is removed approximately three weeks after surgery, the stricture incision site is covered with mucosal epithelial cells without scarring, ensuring good urine flow.

The urethral stricture treatment agent of the present invention has the property of being soluble in ice-cold water, so if it is desired to remove the urethral stricture treatment agent of the present invention for some reason after surgery, it can be easily washed off with ice-cold water.

The following provides examples of the production of "hydrogel-forming polymers" and examples of the present invention to further explain the present invention, but 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 polypropylene oxide-polyethylene oxide copolymer (propylene oxide/ethylene oxide average degree of polymerization about 60/180, Pluronic F-127 manufactured by Asahi Denka Kogyo Co., Ltd.) was dissolved in 30 ml of dry chloroform, and 0.13 g of hexamethylene diisocyanate was added in the presence of phosphorus pentoxide, and the mixture was allowed to react for 6 hours under reflux at the boiling point. After the solvent was distilled off under reduced pressure, the residue was dissolved in distilled water and ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 500,000 to fractionate a high molecular weight polymer and a low molecular weight polymer. The resulting aqueous solution was frozen to obtain an F-127 high polymer and an F-127 low polymer.

1 g of the F-127 high polymer (hydrogel-forming polymer of the present invention, "hydrogel-forming polymer"-1) obtained above was dissolved in 9 g of distilled water under ice cooling to obtain a 10 wt % aqueous solution. The storage modulus of this aqueous solution was measured using a stress-controlled rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz, and was 4 Pa at 10°C, 1890 Pa at 25°C, and 6660 Pa at 37°C. This temperature-dependent change in storage modulus was observed repeatedly and reversibly. On the other hand, when the above F-127 low polymer was dissolved in distilled water at a concentration of 10 wt % below freezing point, it did not gel at all even when heated to 60°C or higher.

Production Example 2
160 moles of ethylene oxide were added to 1 mole of trimethylolpropane by cationic polymerization to obtain a polyethylene oxide triol having an average molecular weight of about 7,000.
100 g of the polyethylene oxide triol obtained above was dissolved in 1000 ml of distilled water, and then filtered at room temperature. 12 g of potassium manganate was gradually added and the mixture was allowed to oxidize 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 polyethylene oxide tricarboxyl.
10 g of the polyethylene oxide tricarboxylate obtained above and 10 g of polypropylene oxide diamino (propylene oxide average degree of polymerization: about 65, Jefferson Chemical Company, USA, product name: Jeffamine D-4000, cloud point: about 9°C) were dissolved in 1000 ml of carbon tetrachloride, 1.2 g of dicyclohexylcarbodiimide was added, and the mixture was allowed to react for 6 hours under reflux at the boiling point. The reaction solution was cooled, solids were removed by filtration, the solvent (carbon tetrachloride) was distilled off under reduced pressure, and the residue was dried in vacuum to obtain a hydrogel-forming polymer of the present invention in which multiple polypropylene oxides and polyethylene oxides are bonded ("hydrogel-forming polymer"-2). 1 g of this polymer was dissolved in 19 g of distilled water under ice-cooling to obtain a 5 wt% aqueous solution. The storage modulus of this aqueous solution was measured using a stress-controlled rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz, and was found to be 1 Pa at 10° C., 550 Pa at 25° C., and 3360 Pa at 37° C. This temperature-dependent change in storage modulus was observed reversibly and repeatedly.

Production Example 3
96 g of N-isopropylacrylamide (manufactured by Eastman Kodak Company), 17 g of N-acryloxysuccinimide (manufactured by Kokusan Chemical Co., Ltd.), and 7 g of n-butyl methacrylate (manufactured by Kanto Chemical Co., Ltd.) were dissolved in 4000 ml of chloroform, and after replacing with nitrogen, 1.5 g of N,N'-azobisisobutyronitrile was added and polymerized at 60°C for 6 hours. The reaction solution was concentrated and then reprecipitated (re-precipitated) in diethyl ether. The solid matter was collected by filtration and then vacuum dried to obtain 78 g of poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate).
Excessive isopropylamine was added to the poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) obtained above to obtain poly(N-isopropylacrylamide-co-n-butyl methacrylate). The cloud point of an aqueous solution of this poly(N-isopropylacrylamide-co-n-butyl methacrylate) was 19° C.

10 g of the poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) and 5 g of aminated polyethylene oxide (molecular weight 6,000, manufactured by Kawaken Fine Chemical Co., Ltd.) were dissolved in 1000 ml of chloroform 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, after which 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 obtain a hydrogel-forming polymer of the present invention in which multiple poly(N-isopropylacrylamide-co-n-butyl methacrylate)s and polyethylene oxides were bonded together ("hydrogel-forming polymer"-3).
1 g of this polymer was dissolved in 9 g of distilled water under ice cooling to obtain an aqueous solution (10 wt%). The storage modulus of this aqueous solution was measured at an applied frequency of 1 Hz using a stress-controlled rheometer (AR500, manufactured by TA Instruments) and was found to be 1 Pa or less at 10°C, 30 Pa at 25°C, and 250 Pa at 37°C. This temperature-dependent change in storage modulus was observed repeatedly and reversibly.

Production Example 4
(Sterilization Method)
2.0 g of the above-mentioned hydrogel-forming polymer of the present invention ("hydrogel-forming polymer"-3) was placed in an EOG (ethylene oxide gas) sterilization bag (manufactured by Hogy Medical Co., Ltd., product name: Hybrid sterilization bag), and EOG was filled into the bag using an EOG sterilization device (Easy Pack, manufactured by Iuchi Seiei-do Co., Ltd.), and the bag was left for a day and night at room temperature. After further leaving it for half a day at 40°C, the EOG was removed from the bag and aeration was performed. The bag was placed in a vacuum dryer (40°C) and sterilized by leaving it for half a day with occasional aeration.
It was separately confirmed that this sterilization procedure did not change the storage modulus of the aqueous polymer solution.

Production Example 5
71.0 g of N-isopropylacrylamide and 4.4 g of n-butyl methacrylate were dissolved in 1117 g of ethanol. An aqueous solution of 22.6 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by Nippon Oil & Fats Co., Ltd.) dissolved in 773 g of water was added to this, and the mixture was humidified to 70°C under a nitrogen stream. While maintaining the temperature at 70°C under a nitrogen stream, 0.8 ml of N,N,N',N'-tetramethylethylenediamine (TEMED) and 8 ml of a 10% aqueous solution of filtered ammonium sulfate (APS) were added and allowed to react mechanically for 30 minutes. Furthermore, 0.8 ml of TEMED and 8 ml of a 10% aqueous solution of APS were added four times at 30-minute intervals to complete the polymerization reaction. After cooling the reaction solution to 10°C or less, 5 L of cooled distilled water at 10°C was added to dilute it, and the mixture was concentrated to 2 L at 10°C using an ultrafiltration membrane with a molecular weight cutoff of 100,000.
The concentrated solution was diluted with 4 L of chilled distilled water, and the above ultrafiltration concentration procedure was carried out again. The above dilution and ultrafiltration concentration procedures were repeated five more times to remove those with molecular weights of 100,000 or less. The material that was not filtered by this ultrafiltration (those remaining in the ultrafiltration membrane) was collected and freeze-dried, yielding 72 g of a hydrogel-forming polymer of the present invention having a molecular weight of 100,000 or more ("hydrogel-forming polymer"-5).

1 g of the hydrogel-forming polymer of the present invention obtained above ("Hydrogel-forming polymer"-5) was dissolved in 9 g of distilled water under ice cooling to obtain a 10 wt % aqueous solution. The storage modulus of this aqueous solution was measured using a stress-controlled rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz, and was found to be less than 1 Pa at 10°C, 80 Pa at 25°C, and 460 Pa at 37°C. This temperature-dependent change in storage modulus was observed repeatedly and reversibly.

Production Example 6
42.0 g of N-isopropylacrylamide and 4.0 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution of 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by Nippon Oil & Fats Co., Ltd.) dissolved in 65.1 g of water was added to the mixture, and the mixture was heated to 70° C. under a nitrogen stream. While maintaining the temperature at 70° C. under a nitrogen stream, 0.4 ml of N,N,N',N'-tetramethylethylenediamine (TENED) and 4 ml of a 10% aqueous solution of ammonium persulfate (APS) were added and reacted at 30-minute intervals. Furthermore, 0.4 ml of TENED and 4 ml of a 10% aqueous solution of APS were added four times at 30-minute intervals to complete the polymerization reaction. After cooling the reaction solution to 59° C. or less, 5 L of cooled distilled water at 5° C. was added to dilute the mixture, and the mixture was concentrated to 2 L at 5° C. using an extraocular filtration membrane with a molecular weight cutoff of 100,000.
The concentrated solution was diluted with 4 L of chilled distilled water, and the above-mentioned extraocular filtration concentration operation was carried out again. The above-mentioned dilution and ultrafiltration concentration operations were repeated five more times to remove those with molecular weights of 100,000 or less. The material that was not filtered by this ultrafiltration (those remaining in the ultrafiltration membrane) was collected and freeze-dried, and 40 g of the hydrogel-forming polymer of the present invention having a molecular weight of 100,000 or more ("hydrogel-forming polymer"-6) was obtained.
1 g of the hydrogel-forming polymer of the present invention obtained above ("hydrogel-forming polymer"-6) was dissolved in 9 g of distilled water under ice cooling to obtain a 10 wt % aqueous solution. The storage modulus of this aqueous solution was measured using a stress-controlled rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz, and was 43 Pa at 10°C, 680 Pa at 25°C, and 1310 Pa at 37°C. This temperature-dependent change in storage modulus was observed repeatedly and reversibly.

Production Example 7
45.5 g of N-isopropylacrylamide and 0.56 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution of 1.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by Nippon Oil & Fats Co., Ltd.) I dissolved in 65.1 g of water was added to this, and heated to 70°C under a nitrogen stream. While maintaining the temperature at 70°C under a nitrogen stream, 0.4 ml of N,N,N',N'-tetramethylethylenediamine (TENED) and 4 ml of a 10% aqueous solution of filtered ammonium sulfate (APS) were added and reacted with stirring for 30 minutes. Furthermore, 4.0 ml of TENED and 4 ml of a 10% aqueous solution of APS were added four times at 30-minute intervals to complete the polymerization reaction. After cooling the reaction solution to below 10°C, 5 L of cooled distilled water at 10°C was added to dilute it, and the solution was concentrated to 2 L at 10°C using an ultrafiltration membrane with a molecular weight cutoff of 100,000.
The concentrated solution was diluted with 4 L of chilled distilled water, and the above ultrafiltration concentration operation was carried out again. The above dilution and ultrafiltration concentration operations were repeated five more times to remove those with molecular weights of 100,000 or less. The material that was not filtered by this ultrafiltration (those remaining in the ultrafiltration membrane) was collected and freeze-dried, yielding 22 g of a hydrogel-forming polymer of the present invention having a molecular weight of 100,000 or more ("hydrogel-forming polymer"-7).
1 g of the hydrogel-forming polymer ("hydrogel-forming polymer"-7) obtained above was dissolved in 9 g of distilled water under ice cooling to obtain a 10 wt%) aqueous solution. The storage modulus of this aqueous solution was measured using a stress-controlled rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz, and was found to be 1 Pa or less at 10°C, 1 Pa or less at 25°C, and 90 Pa at 37°C. This temperature-dependent change in storage modulus was observed repeatedly and reversibly.

Example 1
The freeze-dried hydrogel-forming polymer-6 obtained in Production Example 6 was sterilized with EOG in the same manner as in Production Example 4. The hydrogel-forming polymer-6 after EOG sterilization was dissolved in a phosphate buffer solution at a concentration of 10 wt% under ice cooling. Oral mucosal tissue (2 cm x 1 cm) from a male patient with ureteral stenosis was collected, and the patient's own serum (equivalent amount to the cells) was added to the collagenase-treated cells, and the cells were dispersed in the above-mentioned hydrogel-forming polymer-6 in a phosphate buffer solution under ice cooling. This cell dispersion was heated to 37°C to gel, and cultured for 10 days to increase the number of cells by about 10 times. The patient was incised at the site of urethral stenosis by transurethral endoscopic incision, and a urethral catheter was inserted, and the urethral stenosis therapeutic agent of the present invention after the cell culture, which had been cooled on ice, was injected between the area around the urethral catheter and the incision site on the inner surface of the urethra. The urethral stenosis therapeutic agent of the present invention was warmed by body temperature and immediately gelled, and was placed so as to cover the entire incision site on the inner surface of the urethra. The urethral catheter was removed 3 weeks after surgery. The stricture incision site was covered with mucosal epithelial cells without scarring, and good urinary flow was ensured. Similar treatment was performed on 10 patients, and restenosis was not observed in any of them.

Example 2
Ten patients underwent the same treatment as in Example 1, except that oral mucosal cells and serum from male ureteral stenosis patients were not added. As a result, no restenosis was observed in any of the patients, but healing of the stricture incision site was observed in two cases.

Example 3
Ten patients underwent the same treatment as in Example 2, except that the freeze-dried hydrogel-forming polymer-5 obtained in Production Example 5 was used instead of the freeze-dried hydrogel-forming polymer-6 obtained in Production Example 6. As a result, restenosis was observed in two cases.

Comparative Example 1
Ten patients underwent the same treatment as in Example 1, except that the freeze-dried hydrogel-forming polymer-7 obtained in Production Example 7 was used instead of the freeze-dried hydrogel-forming polymer-6 obtained in Production Example 6. As a result, scarring was observed at the stricture incision site in all patients. It is believed that the storage modulus of the hydrogel-forming polymer-7 at 37°C was low, less than 100 Pa, and therefore it did not function as a therapeutic agent for urethral stricture of the present invention.

Comparative Example 2
In Example 1, the lyophilized hydrogel-forming polymer-6 after EOG sterilization was dissolved in a phosphate buffer solution at a concentration of 11 wt % under ice cooling. The storage modulus of this aqueous solution was measured using a stress-controlled rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz, and was found to be 75 Pa at 10° C. This solution had low fluidity even under ice cooling, and could not be injected into the site of urethral stricture via a catheter, and did not function as a therapeutic agent for urethral stricture of the present invention.

Claims (5)

A pharmaceutical composition for humans, comprising human oral mucosal cells and an equal amount of serum to a thermoreversible hydrogel-forming polymer-containing liquid,
The thermoreversible hydrogel-forming polymer-containing liquid has a storage modulus of 43 Pa at 10° C. , a storage modulus of 680 Pa at 25° C., and a storage modulus of 1310 Pa at 37° C .;
The pharmaceutical composition for humans is for treating urethral stricture and does not contain mitomycin C;
Here, the thermoreversible hydrogel-forming polymer is a thermoreversible hydrogel-forming polymer which is a polymer of N-isopropylacrylamide, n-butyl methacrylate, and polyethylene glycol dimethacrylate.
The pharmaceutical composition for human use. The pharmaceutical composition according to claim 1 , wherein the human oral mucosal cells are autologous cells. A therapeutic agent for treating human urethral stricture, comprising the pharmaceutical composition according to claim 1 or 2 . The therapeutic agent for human urethral stricture according to claim 3 , which is cooled to a temperature of 10° C. or less, injected into the inner surface of the urethra, and left on the inner surface of the urethra. The method for treating human urethral stricture according to claim 4 , wherein the inner surface of the urethra includes a site incised by a transurethral endoscopic technique.