JP7740691B2 - injection material - Google Patents

Description

The present invention relates to an injection material that is injected into living tissue.

EMR (endoscopic mucosal resection) and ESD (endoscopic submucosal dissection) are known procedures in which an injection material is injected under the mucosa of the lesion to elevate and remove the lesion. The main material used in these injection materials is a highly viscous polymeric polysaccharide (see Patent Documents 1 to 3).

As mentioned above, conventional injectable materials rely on the high viscosity of the main material, which makes them difficult to use due to the high resistance they create when injected submucosally. Additionally, the protrusions that form after submucosal injection become hard, which can have an undesirable effect on the muscle layer and can make it difficult to ligate the protrusions by repelling the snare (electrically conductive wire) used in EMR.

Patent No. 4607842 Patent No. 6099044 Japanese Patent Application Laid-Open No. 2001-192336

In light of the above circumstances, the present invention aims to provide an injection material that is low in viscosity, easy to inject, and can adequately maintain the elevated state of biological tissue for the desired period of time.

One aspect of the present invention provides an injection material that is injected into biological tissue. This injection material contains phosphorylated pullulan.

One aspect of the present invention has the advantageous effect of being low-viscosity, easy to inject, and capable of sufficiently maintaining the elevated state of biological tissue for the desired period of time.

1 is a graph showing the results of a viscosity test. 1 is a graph showing the results of an endoscopic puncture needle (tube length: 1,600 mm) passage test. 1 shows photographs showing the results of a test using an isolated porcine esophagus model. 1 is a graph showing the results of a porcine isolated stomach model test. 10 is a photograph showing the progress of EMR when the injection material of Example 4 is used. 10 is a photograph showing the progress of ESD when the injection materials of Example 4 and Comparative Example 1 are used.

The following describes embodiments of the injection material according to the present invention. Various features shown in the following embodiments can be combined with each other.
<Injection material>
The injection material of the present invention is a composition that is injected into living tissue and contains phosphorylated pullulan.
In this specification, the term "phosphorylated pullulan" includes free phosphorylated pullulan and salts of phosphorylated pullulan (for example, sodium salt, potassium salt, etc.).
In the following, a liquid injection material that further contains a solvent will be described.

Examples of biological tissues include mucous membranes, skin, and serous membranes (pleura, peritoneum, pericardium), with mucous membranes being preferred. Most mucosal tissues are difficult to see from outside the body and are located in places that are difficult to treat, but by using the injectable material of the present invention, the lesions in the mucosal tissues can be elevated, making them easier to separate and excise.
Pullulan is a polysaccharide having a structure in which maltotriose units, each of which is formed by α-1,4-bonding three glucose molecules, are repeatedly linked linearly via α-1,6-bonding. The phosphorylated pullulan referred to in the present invention is a phosphorylated form of pullulan. Specific examples include the phosphorylated pullulan disclosed in JP-A-2007-006978, JP-A-2008/010517, JP-A-2009/091001, and JP-A-2011/102530. More preferred examples include high molecular weight pullulan in which the interior of pullulan or multiple pullulan molecules are crosslinked with a phosphorylating agent.
Pullulan is a highly water-soluble polysaccharide, and its aqueous solution exhibits low viscosity. Pullulan is also listed in the Japanese Pharmacopoeia, ensuring its safety.

According to the unique findings of the present inventors, although phosphorylated pullulan is a polymeric compound, its aqueous solution has a significantly lower viscosity than the aqueous solutions of other polymeric polysaccharides, possibly reflecting the properties of pullulan. On the other hand, phosphorylated pullulan has a dendritic structure (dendrimer-like structure) due to cross-linking, and therefore does not easily pass through the gaps between biological tissues even after injection into biological tissues (especially submucosal). Therefore, phosphorylated pullulan is less likely to diffuse and be washed away, and therefore its protruding state can be sufficiently maintained for a desired period of time.
Furthermore, the protuberances created by injecting phosphorylated pullulan under biological tissue have a moderate degree of flexibility, which has the advantage that they can be accurately and reliably ligated using a snare (conductive wire) used in EMR.

The phosphorylating agent used for cross-linking pullulan is not particularly limited, but examples thereof include phosphorus oxychloride (POCl ₃ ), phosphorus trichloride (PCl ₃ ), phosphorus pentachloride (PCl ₅ ), alkyl esters of phosphorus chloride, trisodium trimetaphosphate (STMP), etc. As the phosphorylating agent, one or an appropriate combination of two or more of the above compounds can be used.
Among these, the phosphorylating agent is preferably at least one of phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride, and alkyl esters of phosphorus chloride, and more preferably phosphorus oxychloride. Phosphorylated pullulan obtained using these phosphorylating agents is preferred from the viewpoint of safety.

The number average molecular weight (Mn) of phosphorylated pullulan is not particularly limited, but is generally preferably about 200,000 to 2,000,000, more preferably about 500,000 to 1,600,000, and even more preferably about 800,000 to 1,400,000.
In this specification, the number average molecular weight (Mn) of phosphorylated pullulan can be measured by gel permeation chromatography (GPC) analysis.

The degree of phosphorylation of phosphorylated pullulan can be defined as the proportion of hydroxyl groups substituted with phosphate groups (hydroxyl group substitution ratio) among all hydroxyl groups contained in one molecule of pullulan. The hydroxyl group substitution ratio is usually preferably 1% or more by number, more preferably about 1 to 30% by number, and even more preferably about 5 to 20% by number.
The phosphorus content per molecule of phosphorylated pullulan is usually preferably about 0.1 to 15% by mass, more preferably about 0.1 to 10% by mass.

The proportion of phosphorylated hydroxyl groups in phosphorylated pullulan can be calculated by measuring the phosphorus content by performing elemental analysis of phosphorylated pullulan using inductively coupled plasma (ICP) atomic emission spectrometry, and assuming that all of the measured phosphorus is derived from phosphorylated hydroxyl groups.
The content of phosphate groups can be calculated assuming that all of the phosphorus content is derived from phosphate groups.

The content of phosphorylated pullulan in the injection material is usually preferably about 0.1 to 5% by mass, more preferably about 0.2 to 2.5% by mass, and even more preferably about 0.4 to 1.25% by mass. An injection material containing phosphorylated pullulan in this range can have a lower viscosity.
Specifically, the viscosity of the injection material at a product temperature of 25°C is usually preferably 30 mPa·s or less, more preferably 20 mPa·s or less, even more preferably 10 mPa·s or less, and particularly preferably about 0.1 to 5 mPa·s. The viscosity of the injection material at a product temperature of 25°C is measured using a tuning fork vibrating viscometer under the following conditions: when the viscosity is 1 to 10 mPa·s, the rotation speed is 60 rpm; when the viscosity is 11 to 100 mPa·s, the rotation speed is 6 rpm.

An injection material with such low viscosity can be smoothly and reliably injected into living tissue. Furthermore, because of its excellent storage stability and ease of handling, it is particularly suitable for use under endoscopy. Furthermore, no injection material with such low viscosity has existed to date.
Furthermore, unlike conventional injection materials, this injection material has low viscosity, so when injected under biological tissue, it is less likely to cause damage by pushing aside the muscle layer, and therefore the risk of complications due to muscle layer damage (e.g., delayed gastrointestinal perforation) can be significantly reduced.

When used under an endoscope, the injection material preferably also has the following properties:
Specifically, when the injection material is passed through an endoscopic puncture needle (tube length: 1,600 mm) at a pushing speed of 25 mm/min and a product temperature of 25°C, the pushing force is usually preferably 20 N or less, more preferably 15 N or less, and even more preferably about 0.5 to 10 N. Because such an injection material has low viscosity, it can be easily injected smoothly and reliably into the submucosa under endoscopy with little burden on the practitioner.

From the viewpoint of improving visibility after injection into living tissue, the injection material preferably further contains a coloring agent to be colored.
Although various dyes can be used as colorants, acid dyes are preferred because of their high affinity with phosphorylated pullulan. Acid dyes are less likely to be released during storage and even after injection into living tissue (i.e., the injectable material is less likely to discolor). This improves the visibility of the lesion and facilitates subsequent treatment.

Examples of acid dyes include brilliant blue FCF, indigo carmine, soluble blue OBC, naphthol green B, eosin Y, acid blue black 10B, and guinea green B.
Furthermore, the acid dye is preferably a compound that is resistant to deterioration due to heating during autoclave sterilization of the injection material or exposure to light. From this perspective, brilliant blue FCF is a suitable acid dye.
Furthermore, if the injection material is colored in advance, the process of separately mixing a coloring agent into the injection material at the operating site can be omitted, resulting in high operability and convenience.

The injection material preferably further contains electrolytes and/or non-reducing carbohydrates.
By adding electrolytes and non-reducing carbohydrates to the infusion material, the osmotic pressure (crystalloid osmotic pressure) of the infusion material can be adjusted to approach a physiological state (physiological osmotic pressure), thereby further increasing the safety of the infusion material when administered to a living body (administered under living tissue).
Although the osmotic pressure of the injection material can be adjusted by adding only electrolytes, the inventors have found that adding a large amount of electrolytes can sometimes result in an excessive decrease in the viscosity of the injection material, which is presumably due to the shrinkage of phosphorylated pullulan molecules caused by the influence of the electrolytes.

On the other hand, by adding a predetermined amount of electrolyte to the injection material, the injection material can be made conductive. Such an injection material made conductive can cut well with high-frequency current at the protuberance formed by injecting it under biological tissue. Furthermore, since the injection material of the present invention does not have a high viscosity, bubbling is unlikely to occur during resection. Therefore, the injection material of the present invention is suitable for ESD.
Therefore, there is a preferred range for the amount of electrolyte added to the injection material. Specifically, the content of the electrolyte in the injection material is usually preferably about 0.15 to 0.75 mass %, and more preferably about 0.35 to 0.55 mass %.
The electrolyte is not particularly limited, but examples thereof include sodium chloride, potassium chloride, magnesium chloride, etc. As the electrolyte, one or more of the above compounds can be used in appropriate combination.

By adding non-reducing carbohydrates to the injection material, the osmotic pressure adjustment effect of electrolytes can be complemented, and the osmotic pressure of the injection material can be more accurately adjusted to physiological conditions. Furthermore, the use of non-reducing carbohydrates not only makes the injection material less likely to discolor, but also prevents discoloration due to deterioration or alteration during autoclave sterilization.
Here, non-reducing carbohydrates refer to sugars in which the reducing group of a monosaccharide is bonded to another sugar via a glycosidic bond or the like and therefore does not exhibit reducing activity.
Examples of non-reducing carbohydrates in the present invention include disaccharides such as trehalose and sucrose, trisaccharides such as raffinose, panose, melezitose, gentianose, lactosucrose and kestose, tetrasaccharides such as stachyose and nystose, pentasaccharides such as verbascose, and sugar alcohols. As the non-reducing carbohydrate, one or a suitable combination of two or more of the above-mentioned carbohydrates can be used.

Among these, trehalose is preferred as a non-reducing carbohydrate. Trehalose is also used as a protective substance for proteins, biological tissues, organs, various drugs such as anticancer drugs, and physiologically active substances. However, there have been no reports on the effect of trehalose on phosphorylated pullulan when used in combination with phosphorylated pullulan. Trehalose also has the advantage of being readily available as an endotoxin-free commercial product.
The content of non-reducing carbohydrates in the injection material is preferably about 0.5 to 10% by mass, more preferably about 2.5 to 7.5% by mass, so that the non-reducing carbohydrates can exert multiple effects.
The solvent used to prepare the injection material may contain water as the main component, and may also contain other organic solvents, such as alcohols such as ethanol.

<Method of producing phosphorylated pullulan>
First, pullulan is treated in an aqueous solution of an alkaline compound.
Specifically, pullulan, an alkaline compound, and water are mixed with stirring to prepare an aqueous solution containing pullulan and an alkaline compound.
Examples of the alkaline compound include sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium oxide, sodium carbonate, sodium sulfite, ammonia, etc. As the alkaline compound, one or a suitable combination of two or more of the above compounds can be used, but sodium hydroxide and potassium hydroxide are preferred.

The aqueous solution containing pullulan and an alkaline compound may be prepared by adding pullulan to an alkaline aqueous solution in which the alkaline compound has been dissolved in water and stirring and mixing, or by adding pullulan and the alkaline compound all at once to water and stirring and mixing, or by pre-mixing pullulan and the alkaline compound in a solid state, and then adding the mixture to water and stirring and mixing.
In either case, it is sufficient that pullulan and the alkaline compound are dissolved in the aqueous solution, but it is preferable to continue stirring even after they are dissolved in order to ensure uniform reactivity.

The content (concentration) of the alkaline compound in the aqueous solution is usually preferably 0.1% by mass or more, more preferably about 1 to 50% by mass, and even more preferably about 10 to 30% by mass.
In addition, the mass ratio of the alkali compound to pullulan (alkali compound/pullulan) is preferably about 0.1/1 to 20/1, more preferably about 0.5/1 to 10/1, and even more preferably about 1/1 to 5/1, from the viewpoint of suppressing hydrolysis of pullulan.

From the viewpoint of preventing or suppressing unwanted coloration and hydrolysis of pullulan, the treatment temperature (stirring temperature) in the aqueous solution is usually preferably about −5 to 50° C., more preferably about 0 to 50° C., even more preferably about 0 to 25° C., and particularly preferably about 4 to 25° C.
Furthermore, from the viewpoint of ensuring the progress of the reaction and reducing unreacted materials, the treatment time (stirring time) is usually preferably about 0.01 to 96 hours, more preferably about 0.1 to 48 hours, and even more preferably about 0.1 to 24 hours.

The pH of the aqueous solution during treatment is not particularly limited, but is preferably adjusted to alkaline with an alkaline compound, and is usually more preferably adjusted to 10 or higher, and even more preferably adjusted to 11 to 14.
In this way, pullulan is treated with an alkaline compound.

The thus treated pullulan is then reacted with a phosphorylating agent, a phosphorus compound (for example, phosphorus oxychloride).
For example, the reaction is carried out by adding a phosphorus compound to an aqueous solution containing pullulan and an alkaline compound and mixing the mixture with stirring.

The ratio of the amounts of pullulan and the phosphorus compound can be adjusted appropriately depending on the degree of phosphorylation of the resulting phosphorylated pullulan (the proportion of hydroxyl groups substituted with phosphate groups among all hydroxyl groups contained in one molecule of pullulan).
The phosphorus compound is mixed into an aqueous solution containing pullulan and an alkaline compound. The mixing method may include, for example, a method in which the aqueous solution containing pullulan and an alkaline compound is stirred while the phosphorus compound is added dropwise.

The temperature during mixing is usually preferably about −5 to 50° C., more preferably about 0 to 45° C., even more preferably about 0 to 10° C., and particularly preferably about 4 to 10° C. Mixing at such a low temperature suppresses hydrolysis of pullulan and also prevents unwanted coloration.
Furthermore, by suppressing side reactions at low temperatures, it is possible to prevent crosslinking reactions caused by diesters, etc. By carrying out the reaction at low temperatures, the hydrolysis reaction caused by water is delayed, and as a result, the phosphorylation reaction of pullulan is further promoted.

The above temperature refers to the temperature of the aqueous solution before the phosphorus compound is mixed in. It is preferable to maintain the temperature of the aqueous solution at the above temperature while the phosphorus compound is being mixed in.
The pH of the aqueous solution when the phosphorus compound is mixed is not particularly limited, but since the alkaline compound and pullulan are mixed in advance, it is preferably adjusted to an alkaline pH, more preferably adjusted to 8 or higher.

After the phosphorus compound is mixed, it is preferable to continue stirring the aqueous solution for a predetermined period of time to allow the reaction to proceed, in order to reduce unreacted products.
The predetermined time is usually preferably about 0.1 to 96 hours, more preferably about 0.1 to 48 hours, and even more preferably about 0.1 to 24 hours.

In the production of phosphorylated pullulan as described above, in addition to pullulan, an alkaline compound and a phosphorus compound, synthetic polymers and the like can also be used.
As the synthetic polymer, polyvinyl alcohol is preferred from the viewpoint of having a large number of hydroxyl groups. The content of polyvinyl alcohol in the aqueous solution used in the reaction is usually preferably about 0.01 to 10% by mass, more preferably about 1 to 3% by mass.

In this way, phosphorylated pullulan is obtained.
The phosphorylated pullulan is obtained in a dissolved state in an aqueous solution, but since the reaction is carried out at a low temperature, excessive cross-linking of pullulan by phosphorus compounds is suppressed. Therefore, solid phosphorylated pullulan can be recovered by removing by-products (e.g., sodium phosphate, sodium chloride) from the aqueous solution after the reaction and then removing water.
The by-products can be removed by, for example, fractionation, filtration, dialysis, salting out, or other known purification methods.

Specifically, the aqueous solution obtained after the reaction can be dialyzed using a regenerated cellulose membrane, an ultrafiltration membrane, or the like to remove by-products, or can be further concentrated (purified) by cross-flow filtration using an ultrafiltration membrane.
Furthermore, according to the above-described method for producing phosphorylated pullulan, the proportion of crosslinked pullulan in the pullulan used as the raw material is preferably 0.5% or more, more preferably 1% or more, and even more preferably 2% or more.

As described above, according to this embodiment, it is possible to provide an injection material that has low viscosity and is easy to inject, and that can adequately maintain the protuberance of biological tissue for a desired period of time.
Furthermore, endotoxins can be easily and reliably removed from the phosphorylated pullulan produced as described above by treatment with an alkaline compound. Furthermore, when trehalose is used in combination with the phosphorylated pullulan, commercially available products from which endotoxins have been sufficiently removed are readily available, and such trehalose can be used preferably. Therefore, the injection material of the present invention thus obtained can easily achieve endotoxin-free status.
Therefore, the injection material of the present invention can be suitably used as an endoscopic submucosal injection material in, for example, EMR (endoscopic mucosal resection) and ESD (endoscopic submucosal dissection), which are procedures in which the injection material is injected submucosally into the lesion to lift and excise the lesion.

The injection material of the present invention can be stored in various forms, such as a prefilled syringe, a vial, a bottle, a bag, a tube, and the like.
In the above embodiment, the injection material is described as being liquid and containing a solvent, but the injection material of the present invention may be solid and not contain a solvent.

Furthermore, it may be provided in the following aspects.
The injection material further contains a solvent and is in a liquid state.
The injection material, wherein the content of the phosphorylated pullulan in the injection material is 0.1 to 5% by mass.
The injection material has a viscosity of 30 mPa·s or less at a product temperature of 25°C.
The injection material has a pressing force of 20 N or less when the injection material is passed through an endoscopic puncture needle (tube length: 1,600 mm) at a pushing speed of 25 mm/min and a product temperature of 25°C.
The injection material further comprises an electrolyte and/or a non-reducing carbohydrate.
The injection material further contains a solvent and is in a liquid state, and the content of the electrolyte in the injection material is 0.15 to 0.75 mass %.
The injection material further contains a solvent and is in a liquid state, and the content of the non-reducing carbohydrate in the injection material is 0.5 to 10 mass %.
The injection material, wherein the non-reducing carbohydrate is trehalose.
The injection material further comprises an acid dye.
The injection material, wherein the biological tissue is a mucous membrane.
Of course, this is not the case.

As stated above, various embodiments of the present invention have been described, but these are presented as examples and do not limit the scope of the invention in any way. These novel embodiments may be embodied in a variety of other forms, and various omissions, substitutions, and modifications may be made without departing from the spirit of the invention. These embodiments and their variations are within the scope and spirit of the invention, and are also within the scope of the invention and its equivalents as set forth in the claims.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
1. Preparation of phosphorylated pullulan First, 20 g of pullulan (manufactured by Hayashibara Co., Ltd., number average molecular weight: 300,000-350,000, white) was dissolved in 1.5 L of water together with 19.7 g of sodium hydroxide (an alkaline compound), and the solution was stirred overnight at room temperature (20°C). The pH of the resulting aqueous solution was 14 or higher, and the mass ratio of sodium hydroxide to pullulan was approximately 1.

Thereafter, the aqueous solution was cooled to 0° C., and then 7.7 mL (11.2 g) of phosphorus oxychloride was added dropwise thereto, followed by stirring at 0° C. for 6 hours to carry out the reaction.
After the reaction was completed, the aqueous solution was dialyzed using a regenerated cellulose membrane to remove the by-products sodium phosphate and sodium chloride until the conductivity reached 30 μS or less.
The aqueous solution was then subjected to cross-flow filtration using an ultrafiltration membrane with a molecular weight cutoff of 5,000, concentrated, and then freeze-dried to obtain 20 g of phosphorylated pullulan (yield: 100%, white).

The number average molecular weight of the phosphorylated pullulan was 800,000 to 1,200,000.

2. Production of injection material (Example 1)
First, 125.0 g of Trehalose SG (manufactured by Hayashibara Co., Ltd.) was placed in a 3 L beaker, and water for injection was added thereto until the content reached 625 g, followed by stirring to obtain an aqueous solution.
Next, 11.25 g of sodium chloride was added to the resulting aqueous solution and stirred.

Thereafter, 583.3 g of a 3% by mass aqueous solution of phosphorylated pullulan was added to the above aqueous solution and stirred.
Next, 5.0 g of a 0.1% by mass aqueous solution of Brilliant Blue FCF (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the above aqueous solution, and then water for injection was added until the total volume reached 2,500 g, followed by stirring.
The injection material was produced in this manner, and the contents of phosphorylated pullulan, trehalose SG, sodium chloride, and brilliant blue FCF were 0.7% by mass, 5% by mass, 0.45% by mass, and 0.0002% by mass, respectively.

Example 2
An injection material was produced in the same manner as in Example 1, except that the content of phosphorylated pullulan was changed to 0.5% by mass.
Example 3
An injection material was produced in the same manner as in Example 1, except that the content of phosphorylated pullulan was changed to 1% by mass.
Example 4
An injection material was produced in the same manner as in Example 1, except that the content of phosphorylated pullulan was changed to 1.4% by mass.

Example 5
An injection material was produced in the same manner as in Example 1, except that the content of phosphorylated pullulan was changed to 2% by mass.
(Comparative Example 1)
Mucoup (registered trademark) (manufactured by Boston Scientific) was prepared as the injection material.
(Comparative Example 2)
Liftal K (registered trademark) (manufactured by Kaigen Pharma Co., Ltd.) was prepared as the injection material.

3. Analysis 3-1. Confirmation of the structure of phosphorylated pullulan The structure of phosphorylated pullulan was confirmed by FT-IR (Fourier transform infrared spectroscopy) analysis.
3-2. Number average molecular weight (Mn) of phosphorylated pullulan
The number-average molecular weight of phosphorylated pullulan was calculated by GPC analysis (one TSKgel guard column (6.0 mm I.D. × 40 mm) (manufactured by Tosoh Corporation) connected to one TSKgel GMPWXL (7.8 mm I.D. × 300 mm) (manufactured by Tosoh Corporation), mobile phase: 200 mM aqueous sodium nitrate solution).
3-3. Phosphorylation degree of phosphorylated pullulan (number %, mass %)
The degree of phosphorylation of phosphorylated pullulan was measured and calculated using an ICP emission spectrometer ("VISTA-PRO" manufactured by Seiko Instruments Inc.).

4. Evaluation 4-1. Viscosity Test To prepare a grout for the viscosity test, phosphorylated pullulan with a number average molecular weight of 1,101,240, a phosphorus content of 3.40% by mass, and a hydroxyl group substitution rate of 6.7% by number was used.
The viscosity of the injection material was measured using a tuning fork vibration viscometer (manufactured by A&D Co., Ltd., "SV-1H") at a product temperature of 25°C under the following conditions: when the viscosity was 1 to 10 mPa·s, the rotation speed was 60 rpm; when the viscosity was 11 to 100 mPa·s, the rotation speed was 6 rpm.
In this test, the grouting materials of Examples 1, 2, 3, 5, and Comparative Examples 1 and 2 were used.
FIG. 1 is a graph showing the results of the viscosity test.
As shown in Figure 1, the injection materials of each Example exhibited low viscosity. Furthermore, by adjusting the content of phosphorylated pullulan, the viscosity of the injection material could be further reduced.

4-2. Endoscopic puncture needle (tube length: 1,600 mm) passage test To prepare the injection material for the passage test, phosphorylated pullulan with a number average molecular weight of 986,318, a phosphorus content of 3.42 mass%, and a hydroxyl group substitution rate of 6.7 number% was used.
A syringe containing the injection material or physiological saline was connected to an endoscopic puncture needle (tube length: 1,600 mm) (manufactured by Top Co., Ltd., "Super Grip") and passed through at a pushing speed of 25 mm/min or 50 mm/min at a temperature of 25°C, and the pushing force required at that time was measured.
In this test, the grouting materials of Examples 1, 2, 3, 5 and Comparative Example 1 were used.
FIG. 2 is a graph showing the results of an endoscopic puncture needle (tube length: 1,600 mm) passage test.
As shown in Figure 2, the injection materials of each Example required a smaller pressing force when passing through a catheter than the injection material of Comparative Example 1. Furthermore, by adjusting the content of phosphorylated pullulan, the pressing force could be further reduced.

4-3. Porcine isolated esophagus model test In this test, the same injection material as prepared in "4-2" was used.
2 mL of the injection material was injected submucosally into the excised porcine esophagus via a 25 G endoscopic puncture needle, and the condition of the injection site was visually observed immediately after injection and 30 minutes later.
In this test, the grouting materials of Examples 1, 3, 4, Comparative Examples 1 and 2 were used.
FIG. 3 is a photograph showing the results of a test using an isolated porcine esophagus model.
Immediately after injection, the injection materials of each Example and Comparative Example 1 were able to elevate the injection site to the same extent. However, the injection material of Comparative Example 2 leaked below the muscular layer, as indicated by the thick arrow, whereas none of the injection materials of the present invention shown in Examples 1 to 4 leaked below the muscular layer.
Furthermore, 30 minutes after injection, the swelling maintenance ability of the injection material of Example 4 was clearly superior to that of the injection materials of each of the comparative examples, and the lateral flow was clearly less. Furthermore, the swelling maintenance ability of the injection materials of Examples 1 and 3 was comparable to that of the injection materials of each of the comparative examples.

4-4. Porcine isolated stomach model test In this test, the same injection material as prepared in "4-2" was used.
2 mL of the injectant or saline solution was injected submucosally into the excised porcine stomach via a 25G needle, and the height of the swelling at the injection site of the injectant or saline solution was measured at predetermined time intervals starting immediately after injection.
In this test, the grouting materials of Examples 1, 3, and 5 and Comparative Example 1 were used.
FIG. 4 is a graph showing the results of a porcine isolated stomach model test.
As shown in FIG. 4, the injection materials of each Example and Comparative Example 1 showed a swelling ability at the injection site that was sufficiently higher than that of physiological saline and was comparable to that of the physiological saline.

4-5. Endoscopic EMR in live pigs
In this EMR, the same injection material as prepared in "4-2" was used.
Using an endoscope, the injection materials of Example 4 and Comparative Example 1 were injected into the submucosa of the stomach of a live pig, and EMR was performed.
FIG. 5 is a photograph showing the progress of EMR when the injection material of Example 4 is used.
When the injection material of Comparative Example 1 was used, snare formation was difficult and EMR was impossible. In contrast, as shown in Figure 5, when the injection material of Example 4 was used, snare formation was easy, EMR was possible, and damage to the muscle layer was also prevented.

4-6. Endoscopic ESD in live pigs
In this ESD, the same injection material as prepared in "4-2" was used.
Using an endoscope, the injection materials of Example 4 and Comparative Example 1 were injected into the submucosa of the stomach of a live pig, and ESD was performed.
FIG. 6 is a photograph showing the progress of ESD when the injection materials of Example 4 and Comparative Example 1 were used.
As shown in Figure 6, when the injection material of Comparative Example 1 was used, intense bubbling (see the area indicated by the circular dotted line) occurred during incision of the submucosal layer, whereas when the injection material of Example 4 was used, there was only slight bubbling.

As such, it can be concluded that the injection material of the present invention is far superior to conventionally known injection materials, particularly when used as an injection material for endoscopic EMR and endoscopic ESD.

Claims (11)

An injection material used by submucosal injection,
Contains phosphorylated pullulan,
The viscosity of the injection material at a product temperature of 25°C is 30 mPa·s or less . The injection material according to claim 1,
Furthermore, the injection material contains a solvent and is in a liquid state. The injection material according to claim 1 or 2,
The injection material has a content of the phosphorylated pullulan in the injection material of 0.1 to 5% by mass. The injection material according to any one of claims 1 to 3,
The injection material , wherein the number average molecular weight (Mn) of the phosphorylated pullulan is 200,000 to 2,000,000 . The injection material according to any one of claims 1 to 4,
The injection material is passed through an endoscopic puncture needle (tube length: 1,600 mm) at a pushing speed of 25 mm/min and a product temperature of 25°C with a pushing force of 20 N or less. The injection material according to any one of claims 1 to 5,
The injection material further comprises electrolytes and/or non-reducing carbohydrates. 7. The injection material according to claim 6,
Furthermore, it contains a solvent and is in a liquid state,
The content of the electrolyte in the injection material is 0.15 to 0.75 mass %. The injection material according to claim 6 or 7,
Furthermore, it contains a solvent and is in a liquid state,
The content of the non-reducing carbohydrate in the injection material is 0.5 to 10% by mass. The injection material according to any one of claims 6 to 8,
The injection material, wherein the non-reducing carbohydrate is trehalose. The injection material according to any one of claims 1 to 9,
The injection material further comprises an acid dye having affinity for the phosphorylated pullulan . The injection material according to claim 10 ,
The acid dye is Brilliant Blue FCF .