LIQUID MEDICAL MATERIAL

Abstract

To provide a liquid medical material maintaining a colloid in a more sol form than a solid at normal temperature, having a higher function as a wound dressing material and a hemostatic material than fibrin glue, and being able to be produced safely and inexpensively. A gelatin aqueous solution including calcium at a concentration of 0.2 M or more and 1.0 M or less, and having a concentration of 5% by weight or more and 40% by weight or less, an average molecular weight of 80,000 or more and 120,000 or less, and a molecular weight distribution of 20,000 or more and 300,000 or less, and transglutaminase inducing crosslinking of the gelatin, are included. It is preferable that the calcium has a concentration of 0.2 M or more and 0.7 M or less, the gelatin has a bloom of 160 or more and 250 or less, and the transglutaminase has activity per unit of 36 U/ml to 400 U/ml.

Claims

1. A liquid medical material comprising: a gelatin aqueous solution including calcium at a concentration of 0.2 M or more and 1.0 M or less and having a concentration of 5% by weight or more and 40% by weight or less, an average molecular weight of 80,000 or more and 120,000 or less, and a molecular weight distribution of 20,000 or more and 300,000 or less; and transglutaminase inducing crosslinking of the gelatin.

2. The liquid medical material according to claim 1, wherein the calcium has a concentration of 0.2 M or more and 0.7 M or less, the gelatin has a bloom of 160 or more and 250 or less, the transglutaminase has activity per unit of 36 U/ml to 400 U/ml.

3. The liquid medical material according to claim 1, wherein a rate of a calcium concentration [M] with respect to a gelatin weight concentration [wt %] in the gelatin aqueous solution is 0.005 M/wt % to 0.040 M/wt %.

4. The liquid medical material according to claim 1, further comprising dextran having a DE value of 10 or more and 25 or less, and an average molecular weight of 5000 or more and 25000 or less, in addition to the transglutaminase.

5. The liquid medical material according to claim 1, further comprising dextran having a DE value of 25 or more, and an average molecular weight of 5000 or less, in addition to the transglutaminase.

6. The liquid medical material according to claim 1, wherein the liquid medical material is a tissue adhesive for living organisms, a hemostatic material, a cell preservation solution, an organ preservation solution, an artificial ointment, an alveolar bone reconstruction agent, an anti-adhesion agent for biological tissues, a mucosal protuberance agent, an anti-post-bleeding agent, a wound dressing material, an implant auxiliary material, or an embolization material in endovascular therapy.

7. The liquid medical material according to claim 1, wherein the material is a venous hemostatic material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 is a graph showing a change of viscoelasticity of each of hemostatic materials when samples having different transglutaminase activity in Examples of the present invention.

[0054] FIG. 2 includes (A) a graph illustrating a relation between a DE value and a hemostasis function and (B) a graph illustrating a relation between an average molecular weight and a hemostasis function, when dextran is included in addition to transglutaminase in Examples of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0055] Hereinafter, a liquid medical material of an embodiment of the present invention will be described.

[0056] A liquid medical material in accordance with the embodiment of the present invention includes a gelatin aqueous solution including calcium at a concentration of 0.2 M or more and 1.0 M or less, and having a concentration of 5% by weight or more and 40% by weight or less, an average molecular weight of 80,000 or more and 120,000 or less, and a molecular weight distribution of 20,000 or more and 300,000 or less; and transglutaminase inducing crosslinking of the gelatin.

[0057] It is preferable that the calcium has a concentration of 0.2 M or more and 0.7 M or less, the gelatin has a bloom of 160 or more and 250 or less, the transglutaminase has activity per unit of 36 U/ml to 400 U/ml.

[0058] The calcium preferably includes, for example, calcium chloride, and calcium carbonate, and particularly preferably calcium chloride.

[0059] It is preferable that a rate of a calcium concentration [M] with respect to a gelatin weight concentration [wt %] in the gelatin aqueous solution is 0.005 M/wt % to 0.040 M/wt %.

[0060] It is preferable that dextran having a DE value of 10 or more and 25 or less, and an average molecular weight of 5000 or more and 25000 or less is included in addition to the transglutaminase.

[0061] Furthermore, it is preferable that dextran having a DE value of 25 or more, and an average molecular weight of 5000 or less is included in addition to the transglutaminase.

[0062] It is that the liquid medical material in accordance with the embodiment of present invention includes, for example, a tissue adhesive for living organisms, a hemostatic material, a cell preservation solution, an organ preservation solution, an artificial ointment, an alveolar bone reconstruction agent, an anti-adhesion agent for biological tissues, a mucosal protuberance agent, an anti-post-bleeding agent, a wound dressing material, an implant auxiliary material, or an embolization material in endovascular therapy.

[0063] In particular, it is preferable that the liquid medical material in accordance with the embodiment of present invention is a venous hemostatic material.

[0064] In the present invention, the “blood” may be human blood, or non-human blood.

[0065] The liquid medical material in accordance with the embodiment of the present invention maintains a colloid in a more sol form than a solid at normal temperature, has a higher function as a wound dressing material and a hemostatic material than fibrin glue, and can be produced safely and inexpensively.

[0066] In the liquid medical material of the embodiment of the present invention, a gelatin material is not solidified (gelled) under a condition at 25° C. and around 37° C. in the living organism, and then can be solidified in 300 seconds, preferably 90 seconds, and further preferably 30 seconds when transglutaminase is added.

[0067] In particular, when the liquid medical material is used in blood, solidification occurs at the same or faster rate than in the absence of blood, and a membrane having very high viscoelasticity can be formed.

[0068] When the liquid medical material is used for covering wound, when transglutaminase is mixed, solidification occurs very fast, and a membrane having very high viscoelasticity can be formed.

Example

[0069] Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited thereto. Note here that in the following Examples, “%” means “% by weight.”

(Test 1)

[0070] The following gelatin/calcium aqueous solutions were prepared.

Types of Gelatin:

[0071] A: Acid-treated gelatin having an average molecular weight of 100,000 (distribution of 20,000 to 300,000)
B: Alkali-treated gelatin having an average molecular weight of 100,000 (distribution of 20,000 to 300,000)
C: Gelatin having an average molecular weight of 20,000 (polypeptide)
D: Gelatin having a molecular weight of approximately 100,000 (distribution of 80,000 to 120,000)
Concentration of gelatin: 5 to 40 wt % (5, 10, 20, 30, and 40 wt %)
Calcium concentration: 0.0 to 1.0 M (0, 0.2, 0.4, 0.5, 0.7, and 1.0 M)
(1) Gelatin aqueous solutions of A, B, C, and D, having concentrations of 5, 10, 20, 30, and 40 wt %, were prepared, respectively. Under a condition at 25° C., the gelatin aqueous solutions of A, B, and D were solidified at concentration of 5%. The gelatin aqueous solution of C was not solidified even at 40% and was in a flow state.
(2) Gelatin/calcium aqueous solutions of A, B, and D, having concentrations of gelatin and concentrations of calcium shown in Table 1 were prepared.

[0072] Under a condition at 25° C., a state of each of the gelatin/calcium aqueous solutions was observed, and evaluated for solidification, flow, or high viscosity.

Solidified: A state in which an aqueous solution does not move at all even if a sample is brought down.
Flow: A state in which an aqueous solution starts to move when a sample is brought down.
High viscosity: A state in which an aqueous solution starts to move gradually a few times after a sample is brought down.

TABLE-US-00001 TABLE 1 Calcium concentration [M] A 0 0.2 0.4 0.5 0.7 1 Gelatin  5 Solidified Flow Flow Flow Flow Flow concentration 10 Solidified Flow Flow Flow Flow Flow [wt %] 20 Solidified High Flow Flow Flow Flow viscosity 30 Solidified Solidified High High Flow Flow viscosity viscosity 40 Solidified Solidified Solidified High High High viscosity viscosity viscosity Calcium concentration [M] B 0 0.2 0.4 0.5 0.7 1 Gelatin  5 Solidified Flow Flow Flow Flow Flow concentration 10 Solidified Flow Flow Flow Flow Flow [wt %] 20 Solidified High Flow Flow Flow Flow viscosity 30 Solidified Solidified High High Flow Flow viscosity viscosity 35 Solidified Solidified Solidified High High Flow viscosity viscosity 40 Solidified Solidified Solidified High High High viscosity viscosity viscosity Calcium concentration [M] D 0 0.2 0.4 0.5 0.7 1 Gelatin  5 Solidified Flow Flow Flow Flow Flow concentration 10 Solidified Flow Flow Flow Flow Flow [wt %] 20 Solidified Solidified Flow Flow Flow Flow 30 Solidified Solidified Solidified Solidified Flow Flow 40 Solidified Solidified Solidified Solidified Solidified Solidified

[0073] From the above results, aqueous solutions that are not solidified, that is, aqueous solutions in a state having a high viscosity or in a flow state, at 25° C., are selected. The conditions for selection are as follows.

<Conditions>

[0074] A, B: 0%<suitable condition≤40% gelatin<concentration at which a gelatin aqueous solution is prepared

[0075] 0.2 M calcium≤suitable condition≤1.0 M calcium<concentration at which a calcium aqueous solution is prepared

C: 0%<suitable condition≤40% gelatin<concentration at which a gelatin aqueous solution is prepared,

[0076] 0 M calcium≤suitable condition<concentration at which a calcium aqueous solution is prepared

D: 0%<suitable condition<40% gelatin

[0077] 0.2 M calcium≤suitable condition≤1.0 M calcium<concentration at which a calcium aqueous solution is prepared

(Test 2)

[0078] In order to examine a range of suitable activity of transglutaminase, change of viscoelasticity of hemostatic materials using samples having different transglutaminase activity were measured.

[0079] Evaluation of samples having different transglutaminase activity was carried out using 20% gelatin containing 0.5 M CaCl.sub.2) (bloom: 250, average molecular weight: 100,000). Table 2 shows transglutaminase activity of each sample. FIG. 1 shows measurement results of change of viscoelasticity.

TABLE-US-00002 TABLE 2 Transglutaminase activity Examples (as BSA) [U/ml] Example 1 51.6 Example 2 43.0 Example 3 34.4 Example 4 25.8 Comparative Example 1 17.2

[0080] The results of FIG. 1 show that transglutaminase used for a crosslinking agent of the hemostatic material is preferably transglutaminase having transglutaminase activity [U/ml] of higher than 17.2, and more preferably higher than 34.4.

(Test 3)

[0081] A crosslinking evaluation test of a hemostatic material was carried out using a blood viscoelasticity testing device (product name “Sonoclot” manufactured by Sienco, Inc.). After the addition of transglutaminase, the relative viscoelasticity is increased by crosslinking. The inclination of the increase is defined as K, and is compared.

<Basic Experiment>

[0082] An experiment was carried out using calcium-containing gelatin shown in Table 3 and transglutaminase having relative activity of 51.6 U/ml. The results are shown in right column of Table 3.

TABLE-US-00003 TABLE 3 Inclination (= K) (relative Composition viscoelasticity/time [/min] Example 1 20% (bloom: 250, average 45.9 molecular weight: 100,000) gelatin, 0.5M CaCl.sub.2 Example 2 30% (bloom: 250, average 45.7 molecular weight: 100,000) gelatin, 0.7M CaCl.sub.2 Example 3 20% (bloom: 200, average 32.4 molecular weight: 100,000) gelatin, 0.5M CaCl.sub.2 Comparative 25% (bloom: 300) gelatin, 10.4 Example 1 2M CaCl.sub.2 (prepared according to the existing patent)

[0083] From the results, it is shown that in Comparative Example 1 prepared according to the existing patent, elongation of the initial viscoelasticity is poor, and solidification does not sufficiently occur over time, and that in Examples 1 to 3, elongation of the initial viscoelasticity is good and solidification easily occurs.

[0084] The 20% gelatin/0.2 M calcium aqueous solution (material a) of B of Test 1 was selected, and the crosslinking test by transglutaminase was carried out. The activity and concentration of transglutaminase (TG) used are shown in Table 4.

TABLE-US-00004 TABLE 4 Activity Concentration Name [U/g] [mg/ml] Product name TGA 86 6.0 KS-CT (manufactured by Ajinomoto Co., Inc.) TGB 86 2.0 KS-CT (manufactured by Ajinomoto Co., Inc.) TGC 86 0.6 KS-CT (manufactured by Ajinomoto Co., Inc.)

[0085] To 100 μL of the material a, 50 μL each of TG having concentrations was added, and the obtained product was stirred, and solidification time was measured. The results are shown in Table 5.

[0086] From the results of Table 5, TGA was selected as TG.

TABLE-US-00005 TABLE 5 Concentration Solidification time Name [mg/ml] [min] TGA 6.0 3 TGB 2.0 6 TGC 0.6 15

[0087] A crosslinking test was carried out using transglutaminase and various gelatin/calcium aqueous solutions. Conditions of the samples subjected to the test are shown in Tables 6 and 7.

[0088] Furthermore, for comparison, Reference Examples were prepared as follows based on Patent Literature 4, and similarly, the cross-linking test was carried out.

[0089] Reference Example 1: A solution was prepared by mixing a 25% (w/w) gelatin solution including 2 M urea, 1 M calcium, and 0.1 M sodium acetate (the gelatin is pig acid-treated gelatin and has a bloom of 270) with 7.5% (w/w) microbial transglutaminase (10% w/w mTG-ACTIVA-TG manufactured by Ajinomoto Co., Inc.) solution including 0.5 M sodium acetate and calcium-independent microbial transglutaminase (mTG).

[0090] Reference Example 2: A solution was prepared using 0.5M sodium citrate instead of 0.5 M sodium acetate in Reference Example 1.

TABLE-US-00006 TABLE 6 Calcium Sample B-system concentration Solidification Surgical No. [%] [M] time [sec] field Examples 1 5 0.2 90 Open Example 2 0.4 420 Co. Example 3 0.5 450 Co. Example 4 0.7 460 Co. Example 5 10 0.2 *) Co. Example 6 0.4 80 Open Example 7 0.5 200 Catheter Example 8 0.7 350 Co. Example 9 20 0.3 30 Open Example 10 0.4 25 Open Example 11 0.5 60 Open Example 12 0.7 350 Co. Example 13 30 0.2 N.D Co. Example 14 0.4 40 Open Example 15 0.5 50 Open Example 16 0.7 200 Catheter Example 17 35 0.2 N.D Co. Example 18 0.4 N.D Co. Example 19 0.5 50 Open Example 20 0.7 140 Catheter Example Re. 150 Co. Example Example 1 Re. 210 Co. Example Example 2 *): A flow state became heterogeneous immediately after transglutaminase was added. N.D.: A state is solid and cannot be mixed.

TABLE-US-00007 TABLE 7 Calcium 20% concentration Solidification Sample No. gelatin Bloom [M] time [sec] Surgical field Examples 21 A-system 200 0.2  40 Open Example 22 0.4  30 Open Example 23 0.5 100 Catheter Example 24 0.7 350 Co. Example 25 B-system 160 0.2  75 Open Example 26 0.4  65 Open Example 27 0.5 100 Catheter Example 28 0.7 420 Co. Example 29 200 0.2  55 Open Example 30 0.4  25 Open Example 31 0.5  40 Open Example 32 0.7 350 Co. Example 33 250 0.2  45 Open Example 34 0.4  15 Open Example 35 0.5  85 Open Example 36 0.7 250 Catheter Example 37 D-system 200 0.2 *) Co. Example 38 0.4 *) Co. Example 39 0.5 *) Co. Example 40 0.7 150 Catheter Example *): A flow state became heterogeneous immediately after transglutaminase was added.

[0091] A liquid medical material preferably has functions that under a condition at 25° C., the liquid medical material is not solidified before crosslinking, does not form a heterogeneous lump even when mixed, and has solidification time after crosslinking (the time at which a viscosity becomes the square or more of its original state (for example, 10 cps.fwdarw.100 cps) of 300 seconds or less, more preferably 90 seconds or less, and particularly 30 seconds or less.

[0092] It is considered to be difficult to use materials that are not solidified under a condition at 25° C. and not solidified in 85 seconds in an open surgical field. Furthermore, it is considered to be difficult to use materials that are not solidified in 300 seconds in a catheter surgical field.

[0093] When 100 μL of blood, 100 μL of 20% gelatin/0.2 M calcium aqueous solution of B (material a), and 50 μL of transglutaminase are mixed, a solidification time was measured. Conditions of samples subjected to the test are shown in Table 8.

TABLE-US-00008 TABLE 8 Calcium 20% concentration Solidification Sample No. gelatin Bloom [M] time [sec] Surgical field Examples 41 A-system 200 0.2 N.D. Co. Example 42 0.4  24 Open Example 43 0.5  80 Catheter Example 44 0.7 280 Catheter Example 45 B -system 160 0.2 N.D. Co. Example 46 0.4  52 Open Example 47 0.5  95 Catheter Example 48 0.7 360 Co. Example 49 200 0.2 N.D. Co. Example 50 0.4  22 Open Example 51 0.5  33 Open Example 52 0.7 280 Catheter Example 53 250 0.2 N.D. Co. Example 54 0.4  12 Open Example 55 0.5  15 Open Example 56 0.7 220 Catheter Example 57 D-system 200 0.2 N.D. Co. Example 58 0.4 N.D. Co. Example 59 0.5 N.D. Co. Example 60 0.7 120 Catheter Example N.D.: When blood and transglutaminase were mixed with material a at 25° C., α state became heterogeneous.

[0094] As shown in Table 8, it is considered to be difficult to use a material, which is not solidified under a condition at 25° C. and not solidified in 60 seconds, for hemostasis, in particular, arterial hemostasis in an open surgical field. Furthermore, it is considered to be difficult to use a material that is not solidified for 200 seconds for hemostasis in a catheter surgical field.

(Test 4)

[0095] The following gelatin/calcium aqueous solution was prepared. An alkali-treated gelatin (concentration of gelatin: 20 wt %)/0.5 M calcium aqueous solution having an average molecular weight of 100,000 (distribution: 20,000 to 300,000). A cross-linking test using transglutaminase was carried out using this gelatin aqueous solution.

[0096] As transglutaminase (TG), transglutaminase (Name: TGA, product name: KS-OT (manufactured by Ajinomoto, Inc.)) having activity of 86 U/g, concentration of 6 mg/ml, and polysaccharide concentration of 600 mg/ml was used. Included polysaccharide was removed, and then the following five types of dextran were added at 600 mg/ml, respectively. Results of the cross-linking test were shown in right part, respectively.

(1) Dextran having a DE value of 38, an average molecular weight of 4000, and being porous: cross-linking reaction occurred within 5 minutes
(2) Dextran having a DE value of 16, an average molecular weight of 15000, and being porous: cross-linking reaction occurred within 30 seconds
(3) Dextran having a DE value of 10, an average molecular weight of 25000, and being porous: cross-linking reaction occurred
(4) Dextran having a DE value of 10, an average molecular weight of 23000, and not being porous: cross-linking test did not occur
(5) Dextran having a DE value of 12, an average molecular weight of 20000, and not being porous: cross-linking test did not occur

[0097] Furthermore, for the hemostasis function, a case where the cross-linking test was not carried out is evaluated as 1, a case where the cross-linking reaction occurred is evaluated as 2, a case where the cross-linking reaction occurred within 5 minutes is evaluated as 3, a case where the cross-linking reaction occurred within 2 minutes is evaluated as 4, and a case where the cross-linking reaction occurred within 30 seconds is evaluated as 5. The relation between the DE value and the hemostasis function is shown in FIG. 2(A), and the relation between the average molecular weight and the hemostasis function is shown in FIG. 2(B).

[0098] From the above results, from the viewpoint of the hemostasis function, it is shown to be preferable that dextran having a DE value of 10 or more and 25 or less, and an average molecular weight of more than 5000 and 25000 or less is included in addition to transglutaminase. Furthermore, it is shown to be preferable that dextran having a DE value of 25 or more and an average molecular weight of 5000 or less is included in addition to transglutaminase.