JP6058367B2 - Implant coating agent, implant and method for producing implant - Google Patents

Description

  The present invention relates to an implant coating agent, an implant, and a method for producing the implant.

  Conventionally, implants are frequently used in the medical and dental fields. As a material of such an implant (implant material), a metal material {titanium, titanium alloy, cobalt chromium alloy, stainless steel, tantalum, vitarium, etc.} (Patent Document 1) or a polymer material {MPC (2-methacryloyloxyethyl) Phosphorylcholine) polymer and polyvinyl alcohol etc.} (Patent Document 2) and ceramics {hydroxyapatite etc.} are generally used. However, these implant materials have a problem that inflammation occurs due to an immune reaction in the body when implanted in a living body.

Japanese Patent No. 4,907,643 International Publication No. 2009/081870

  An object of this invention is to provide the coating agent for implants which can reduce the inflammation by an implant material.

The present inventor has reached the present invention as a result of extensive research.
That is, the present invention is an implant coating agent comprising an artificial protein (A),
(A) has a polypeptide chain (Y) having 2 to 200 consecutive GVGVP sequences (1 ) as the amino acid sequence (X) and / or the following polypeptide chain (Y ′), Y) and (Y ') total number of is 1 to 100, (hydrophobic degree 0.2-1.2 der of a) Ri, (a) is (GAGAGS) ₄ sequence (14) and and / or (GAGAGS) ₂ sequence _{(15), (GVGVP) 4} GKGVP (GVGVP) 3 sequence (19) or (GVGVP) ₄ GKGVP (GVGVP) ₁₁ sequence (20) and a Ru der engineered proteins implants having Implant coated on the surface with the coating agent for implant; surface of the implant material (C1) with a solution (B1) having a concentration of the coating agent for implant of 5 to 100 μg / ml A method for producing an implant comprising a step of coating the surface of an implant material (C1) by at least one method selected from the group consisting of spraying, dipping and coating; the concentration of the coating agent for implants is 5 to 100 μg / ml It is a manufacturing method of an implant including the process of applying a voltage between an implant material (C2) and an electrode in a solution (B2).
Polypeptide chain (Y ′): A polypeptide chain in which 0.1 to 10% of the number of amino acids in (Y) is substituted with lysine and / or arginine.
Implant material (C1): Implant material made of a polymer material and / or a ceramic material.
Implant material (C2): Implant material made of a metal material and / or an ion-bonding ceramic material.

  The implant coating agent of the present invention can reduce inflammation caused by the implant material.

  In the present invention, the artificial protein (A) is artificially produced in order to eliminate animal-derived components, and includes organic synthesis methods (enzymatic methods, solid phase synthesis methods, liquid phase synthesis methods, etc.) and genes. It can be produced by a recombinant method. For organic synthesis methods, see "Biochemistry Experiment Course 1, Protein Chemistry IV (July 1, 1981, edited by the Japanese Biochemical Society, published by Tokyo Chemical Co., Ltd.)" or "Second Biochemistry Experiment Course 2, Protein Chemistry" (Lower) (May 20, 1987, edited by the Japanese Biochemical Society, published by Tokyo Chemical Co., Ltd.), etc. Regarding the gene recombination method, the method described in Japanese Patent No. 3338441 can be applied. Although both the organic synthesis method and the gene recombination method can produce the artificial protein (A), the gene recombination method is preferable from the viewpoint that the amino acid sequence can be easily changed and mass production can be performed at low cost.

The implant coating agent of the present invention is an implant coating agent comprising an artificial protein (A), wherein (A) is a GVGVP array (1), a PGVGV array (2), a VPGVG array (3), a GVPPGV array ( 4), a polypeptide chain (Y) in which any one amino acid sequence (X) of VGVPG sequence (5), GPP sequence, GAP sequence and GAHGGPGPK sequence (6) is continuous, and / or the following poly It has a peptide chain (Y ′), the total number of (Y) and (Y ′) in (A) is 1 to 100, and the hydrophobicity of (A) is 0.2 to 1.2. It is a coating agent for implants.
Polypeptide chain (Y ′): A polypeptide chain in which 0.1 to 10% of amino acids in (Y) are substituted with lysine and / or arginine.

In the present invention, the polypeptide chain (Y) specifically comprises (GVGVP) _a sequence (Y1), (PGVGV) _b sequence (Y2), (VPGVG) _c sequence (Y3), (GVPPGV) _d sequence (Y4 ), (VGVPG) _e sequence (Y5), (GPP) _f sequence (Y6), (GAP) _g sequence (Y7) and polypeptide chain represented by (GAHGPAGPK) _h sequence (Y8). (Here, a to h are the consecutive numbers of the amino acid sequence (X), respectively, and are integers of 2 to 200.)
When the artificial protein (A) has a plurality of polypeptide chains (Y) in one molecule, it may have one kind of the polypeptide chain or two or more kinds.
When the amino acid sequence (X) has a plurality of polypeptide chains (Y) of the same type in the artificial protein (A), the number of consecutive (X) s may be the same or different for each (Y). Also good. That is, a to h may have a plurality of the same polypeptide chains (Y), or a to h may have a plurality of different polypeptide chains (Y).

The amino acid sequence (X) constituting the polypeptide chain (Y) includes GVGVP sequence (1), PGVGV sequence (2), VPGVG sequence (3), and GVPPGV sequence from the viewpoint of affinity with cells and suppression of inflammation. (4) and VGVPG sequence (5) are preferred, and GVGVP sequence (1) and VPGVG sequence (3) are more preferred.
The polypeptide chain (Y) includes (GVGVP) _b sequence (Y1), (PGVGV) _b sequence (Y2), (VPGVG) _c sequence (Y3), (from the viewpoint of affinity with cells and suppression of inflammation. GVPGV) _d sequence (Y4) and (VGVPG) _e sequence (Y5) are preferred, more preferably (GVGVP) _b sequence (Y1) and (VPGVG) _c sequence (Y3).

In the present invention, the polypeptide chain (Y ′) is a polypeptide chain in which 0.1 to 10% of the total number of amino acids in (Y) is substituted with lysine and / or arginine. As a specific example, part or all of the amino acid sequence (X) constituting the polypeptide chain (Y) is substituted with the following amino acid sequence (X ′), and 0.1 to 10 of all amino acids in (Y). Polypeptide chains in which% is substituted with lysine (K) and / or arginine (R).
Amino acid sequence (X ′): An amino acid sequence in which 10 to 80% of the total number of amino acids in the amino acid sequence (X) is substituted with K and / or R.

In the amino acid sequence (X ′), the number of amino acid substitutions in the amino acid sequence (X) (the number substituted with K and / or R) is the inflammation suppression and solubility of (A) (especially solubility in water). ) Is preferably 1 to 5, more preferably 1 to 3.
In addition, in the amino acid sequence (X ′), from the viewpoint of inflammation suppression and solubility of (A) (particularly solubility in water), 10 to 60% of all amino acids in the amino acid sequence (X) are K and / or An amino acid sequence substituted with R is preferred, and more preferably 20 to 60% is an amino acid sequence substituted with K and / or R.
As the amino acid sequence (X ′), from the viewpoint of inflammation suppression, the GKGVP sequence (7), the GVGKP sequence (8), the GKGKP sequence (9), the KPGVG sequence (10), the VPGKG sequence (11) and the KPGKG sequence ( At least one sequence selected from the group consisting of 12) is preferred, and at least one sequence selected from the group consisting of the GKGVP sequence (7) and the KPGVG sequence (10) is more preferred.

  Since the artificial protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y ′), it has high affinity with cells and high biocompatibility. Therefore, by coating the implant material with the coating agent for implants comprising the artificial protein (A) of the present invention, inflammation that has occurred in conventional implants can be suppressed.

Whether or not it is a polypeptide chain (Y ′) is determined when the K and R in the sequence of the artificial protein (A) are replaced with other amino acids (G, A, V, P or H). Judgment is made based on whether or not (Y).
In the polypeptide chain (Y ′), the total number of amino acids substituted with lysine and / or arginine in (Y) is determined from the viewpoints of inflammation suppression and solubility of (A) (particularly solubility in water). The number is preferably 2 to 50, more preferably 3 to 26.
In addition, in the polypeptide chain (Y ′), 0.1-10% of the total number of amino acids in (Y) is a polypeptide chain substituted with lysine and / or arginine. ) Solubility (particularly solubility in water) is preferably 0.5 to 5%, and more preferably 1 to 3%.

  In the present invention, the artificial protein (A) has a polypeptide chain (Y) and / or a polypeptide chain (Y ′), and the total number of (Y) and (Y ′) in (A) is 1. There are ~ 100 artificial proteins. When (A) has (Y) with different types and / or consecutive numbers of (X), each is counted as one, and the number of (Y) is the total. The same applies to (Y ′).

  The artificial protein (A) has (A) a total of 1 to 100 polypeptide chains (Y) and / or polypeptide chains (Y ′) in one molecule, but suppresses inflammation and dissolves (A). From the viewpoint of property (particularly solubility in water), 50 to 100 are preferable, and 75 to 100 are particularly preferable.

In the present invention, the hydrophobicity of the artificial protein (A) is 0.2 to 1.2, but from the viewpoint of inflammation suppression and solubility of (A) (especially solubility in water), 0.5 to 1.2 is preferable, more preferably 0.5 to 0.8, next more preferably 0.55 to 0.75, and particularly preferably 0.6 to 0.72.
The degree of hydrophobicity in (A) indicates the degree of hydrophobicity of the molecule (A). (A) The number of amino acids constituting the molecule (Mα), the degree of hydrophobicity of each amino acid (Nα) And (A) The total number of amino acids in one molecule can be calculated by applying the following formula. The hydrophobicity of each amino acid is determined according to non-patent literature (Albert L. Leninger, David L. Nelson, Reininger's Shinsei Kagaku, Yodogawa Shoten, September 2010, p. 346-347). The following numerical values described in) are used.
Hydrophobicity = Σ (Mα × Nα) / (M _T )
Mα: (A) Number of each amino acid in one molecule Nα: Hydrophobicity of each amino acid M _T : (A) Total number of amino acids in one molecule A (alanine): 1.8
R (arginine): −4.5
N (asparagine): -3.5
D (aspartic acid): -3.5
C (cysteine): 2.5
Q (glutamine): −3.5
E (glutamic acid): −3.5
G (glycine): -0.4
H (histidine): -3.2
I (isoleucine): 4.5
L (leucine): 3.8
K (lysine): -3.9
M (methionine): 1.9
F (phenylalanine): 2.8
P (proline): -1.6
S (serine): -0.8
T (threonine): -0.7
W (tryptophan): -0.9
Y (tyrosine): -1.3
V (valine): 4.2
For example, when (A) is (GVGVP) ₄ GKGVP (GVGVP) ₃ sequence (19), the degree of hydrophobicity in (A) = {16 (number of G) × (−0.4) +15 (number of V ) × 4.2 + 8 (number of P) × (−1.6) +1 (number of K) × (−3.9)} / 40 (total number of amino acids) = 1.00.

In the present invention, it has the polypeptide chain (Y) and / or the polypeptide chain (Y ′), and the hydrophobicity of (A) is within the above range, so that it can be adsorbed on the surface of the implant material. In addition, the affinity with cells is improved.
When (A) has the sequence (X), the affinity with cells is high, but the hydrophobicity is outside the above range, and the coating efficiency on the implant material surface may be reduced. In that case, the degree of hydrophobicity can be reduced by substituting the amino acid in the amino acid sequence (X) with K and R at a ratio of 10 to 80%, and the affinity with cells and coating on the surface of the implant material can be reduced. It is useful as a coating agent for implants that improves the balance with efficiency and suppresses inflammation of the implant.

In the present invention, the artificial protein (A) preferably further has a GAGAGS sequence (13) from the viewpoint of coating efficiency and heat stability. When (A) has a GAGAGS sequence (13), the GAGAGS sequence (13) is 2 to 2 from the viewpoint of coating efficiency, solubility of (A) (especially solubility in water) and stability to heat. It is preferable to have 100 polypeptide chains (S) linked in series.
In the polypeptide chain (S), the number of consecutive GAGAGS sequences (13) is 2 to 100 from the viewpoint of coating efficiency, solubility of (A) (especially solubility in water) and stability to heat. The number is preferably 2 to 80, more preferably 2 to 80, and particularly preferably 2 to 60.
In (A), when having a polypeptide chain (S), (A) one or more (S) may be present in one molecule, but coating efficiency, solubility of (A) (especially solubility in water) 2) to 100, more preferably 10 to 60, from the viewpoint of the stability to heat) and heat stability.

  When the artificial protein (A) has a total of two or more polypeptide chains (Y), polypeptide chains (Y ′) and polypeptide chains (S), it is interposed between the polypeptide chains and the polypeptide chains. It may have an amino acid sequence (Z). (Z) is a peptide sequence in which one or more amino acids are bound, and is not (Y), (Y ′) or (S). The number of amino acids constituting (Z) is preferably from 1 to 10, more preferably from 1 to 5, particularly preferably from the viewpoint of the solubility of (A) (especially solubility in water) and heat stability. The number is preferably 1 to 3. Specific examples of (Z) include VAAGY sequence (16), GAAGY sequence (17), and LGP sequence.

  Each of the polypeptide chain (Y), polypeptide chain (Y ′) and polypeptide chain (S) at both ends in the artificial protein (A) has a terminal amino acid sequence (T) at the N and / or C terminus. You can do it. (T) is a peptide sequence in which one or more amino acids are bound, and is not (Y), (Y ′) or (S). The number of amino acids constituting (T) is preferably 1 to 100, more preferably 5 to 40, particularly from the viewpoint of the solubility (particularly water solubility) of (A) and the stability to heat. The number is preferably 10 to 35. Specific examples of (T) include MDPVVLQRRDWENPGVTQLNRLAAHPPPFASDPM sequence (18) and the like.

In addition to the above (T), the artificial protein (A) is a protein having a special amino acid sequence at the N and / or C terminus of (A) in order to facilitate purification or detection of expressed (A) or Peptides (hereinafter referred to as “purification tags”) may be included. As the purification tag, an affinity purification tag is used. Such purification tags include polyhistidine 6 × His tag, V5 tag, Xpress tag, AU1 tag, T7 tag, VSV-G tag, DDDDK tag, S tag, CruzTag09 ^™ , CruzTag22 ^™ , CruzTag41 ^™ , Glu. -Glu tag, Ha. 11 tag, KT3 tag, maltose binding protein, HQ tag, Myc tag, HA tag, FLAG tag and the like.
An example of a combination of each purification tag (i) and a ligand (ii) that recognizes and binds the tag is shown below.
(I-1) glutathione-S-transferase (GTS) (ii-1) glutathione (i-2) maltose binding protein (MBP) (ii-2) amylose (i-3) HQ tag (ii-3) nickel ( i-4) Myc tag (ii-4) Anti-Myc antibody (i-5) HA tag (ii-5) Anti-HA antibody (i-6) FLAG tag (ii-6) Anti-FLAG antibody (i-7) 6 XHis tag (ii-7) Nickel or cobalt As a method for introducing the purified tag sequence, the nucleic acid encoding the purified tag is inserted into the 5 ′ or 3 ′ end of the nucleic acid encoding the artificial protein (A) in the expression vector. And a method using a commercially available purification tag introduction vector.

  The total content (% by weight) of the polypeptide chain (Y) and the polypeptide chain (Y ′) in one molecule of the artificial protein (A) is the inflammation suppression and solubility of (A) (especially solubility in water). In view of the above, it is preferably 40 to 90% by weight, more preferably 50 to 87% by weight, based on the molecular weight of (A).

The total content of the polypeptide chains (Y) and (Y ′) in the artificial protein (A) can be determined by amino acid sequencing. Specifically, it can be determined by the following measurement method.
<Measuring method of total content of polypeptide chains (Y) and (Y ′)>
The amino acid sequence is determined using a peptide sequencer (protein sequencer) PPSQ-33A manufactured by Shimadzu Corporation. From the determined amino acid sequence, the total content (% by weight) of the polypeptide chains (Y) and (Y ′) is determined by the following mathematical formula (1).
Total content (% by weight) of polypeptide chains (Y) and (Y ′) = Σ (γ × β) / Σ (α × β) × 100 (1)
α: number of each amino acid in the artificial protein (A) β: molecular weight of each amino acid γ: number of each amino acid in the polypeptide chains (Y) and (Y ′)

  The total content (% by weight) of the amino acid sequence (X) and amino acid sequence (X ′) in one molecule of the artificial protein (A) is the viewpoint of inflammation suppression and solubility of (A) (especially solubility in water). From 40 to 90% by weight based on the molecular weight of (A) is preferable, and more preferably 50 to 87% by weight.

  Protein (A) in one molecule of the number of GAGAGS sequences (13) and the total number of amino acid sequences (X) and amino acid sequences (X ′) constituting polypeptide chain (Y) and polypeptide chain (Y ′) The ratio (number of GAGAGS sequences (13): total number of amino acid sequences (X) and amino acid sequences (X ′) constituting polypeptide chains (Y) and (Y ′)) is coating efficiency, dissolution of (A) From the viewpoint of the property (particularly solubility in water) and stability to heat, 1: 2 to 1:20 is preferable, and 1: 2 to 1:10 is more preferable.

  In the present invention, when (A) has the GAGAGS sequence (13), the stability to heat is further improved. In particular, since the ratio of GAGAGS sequence (13) to amino acid sequences (X) and (X ′) is 1: 2 to 1:20, the stability to heat and solubility (especially solubility in water) are improved. Balanced.

The molecular mass of the artificial protein (A) is preferably 15 to 200 kDa, more preferably 60 to 80 kDa, from the viewpoints of solubility (particularly solubility in water) and stability (biodegradability). Within this range, the time until (A) is decomposed is appropriate.
The molecular mass of the artificial protein (A) is determined by a method in which a measurement sample is separated by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and the migration distance is compared with a standard substance.

A part of preferable artificial protein (A) is illustrated below.
(1) Artificial protein having an amino acid sequence (X) of GVGVP sequence (1) (A1-1)
GVGVP sequence (1) is an artificial protein (A1) having a polypeptide chain (Y′1) in which one amino acid in a continuous polypeptide chain (Y1) is substituted with K (lysine), more preferably (GVGVP) ₄ GKGVP (GVGVP) ₃ Artificial protein having a polypeptide chain (Y′1-1) which is the sequence (19) and a polypeptide chain (S1-1) which is the (GAGAGS) ₄ sequence (14) ( A1-1), an artificial protein (A1-2) having a polypeptide chain (S1-2) which is a polypeptide chain (Y′1-1) and (GAGAGS) ₂ sequence (15), and a polypeptide chain (Y '1-1) An artificial protein (A1-3) having a polypeptide chain (S1-1) and a polypeptide chain (S1-2).
Specifically, 12 polypeptide chains (S1-1) of 4 GAGAGS sequences (13) (GAGAGS) ₄ sequences (14) and 8 polypeptide chains (1) of GVGVP sequence (1) ( Y1-1) has 13 (GVGVP) ₄ GKGVP (GVGVP) ₃ sequences (19) (Y′1-1) in which one of V (valine) in Y1-1) is substituted with K (lysine), The molecular mass having a structure in which one polypeptide chain (S1-2) of (GAGAGS) ₂ sequence (15) in which two GAGAGS sequences (13) are consecutively bonded is chemically bonded to An artificial protein (SELP8K, hydrophobicity 0.618) having a sequence (21) of about 70 kDa; a polypeptide chain (S1-2) of (GAGAGS) ₂ sequence (15) having two consecutive GAGAGS sequences (13) and ( G GVP) ₄ GKGVP (GVGVP) ₃ have respectively 17 polypeptide chain (Y'1-1) sequence (19), a molecular mass of about 77kDa sequences having the structure that they become chemically bonded alternately ( 22) artificial protein (SELP0K, hydrophobicity 0.718) and the like.
16 polypeptide chains (S1-2) of GAGAGS ₂ sequence (15) having two consecutive GAGAGS sequences (13) and 16 polypeptide chains (Y1-1) of GVGVP sequence (1) (GVGVP) ₄ GKGVP (GVGVP) ₁₁ sequence (20) (Y′1-2) in which one of the V (valine) in the inside is replaced with K (lysine), and {(S1 -2) (Y'1-2) (S1-2) } 16 by a chemical bond and molecular mass comprising the artificial protein (SELP415K the sequence of about 71 kDa (23), a degree of hydrophobicity 0.693), and the like.
In a polypeptide chain (Y1-1) in which 6 polypeptide chains (S1-2) of the sequence (GAGAGS) ₂ sequence (15) having 2 consecutive GAGAGS sequences (13) and 16 sequences of the GVGVP sequence (1) are present (GVGVP) ₄ GKGVP (GVGVP) ₁₁ sequence (20) (Y′1-2) 6 and GAGAGS sequence (13) 4 An artificial protein (SELP815K) having 6 polypeptide chains (S1-1) having ₄ consecutive (GAGAGS) ₄ sequences (14), and having a molecular mass of about 65 kDa obtained by alternately chemically bonding them (SELP815K) , Hydrophobicity 0.607) and the like.

The coating agent for implants of this invention consists of the said artificial protein (A).
By using the implant coating agent of the present invention, it is possible to provide an implant that can be coated with an implant material and hardly cause inflammation even when implanted in the body.
Examples of implant materials that can be coated with the coating agent of the present invention include metal materials {titanium, stainless steel, zirconium, tantalum, platinum, gold, vitalium and composite materials having two or more of these}, polymer materials {MPC (2-methacryloyloxyethyl phosphorylcholine) polymer and PVA (polyvinyl alcohol), polystyrene, polyethylene, polypropylene and the like} and ceramics {alumina, zirconia, anatase, rutile, hydroxyapatite, etc.} and the like.

  Examples of the method for coating an implant material with the coating agent of the present invention include a step of coating with an implant coating agent in the method for producing an implant of the present invention described later.

  The implant of the present invention is an implant whose surface is coated with the above-described implant coating agent.

The covering area ratio ((N) / (M)) representing the area (N) for fixing the implant coating agent to the surface area (M) of the implant of the present invention is 50 to 100% from the viewpoint of suppressing inflammation. Is preferable, and 80 to 100% is particularly preferable.
The binding amount, inflammation suppression point of view, is preferably 1~1,000ng / mm ^2, particularly preferably from 5~1,000ng / mm ^2.

The binding amount of the implant coating agent in the implant of the present invention is a value measured by the following measurement method.
<Measurement of binding amount>
A mixed solution (C) of Reagent A solution: Reagent B solution: Reagent C solution = 25: 24: 1 attached to Micro BCA ^™ protein assay kit (manufactured by THERMO Fisher Scientific) is prepared. Add the mixture (C) at a rate of 312.5 μL / cm ² to the surface area of the part to be measured for the binding amount of the coating agent for the implant in the implant, and evenly adhere to the surface of the part to be measured at 37 ° C. for 2 hours. Leave still.
After 2 hours of standing, the chelate production amount of bicinchoninic acid (BCA) and Cu ⁺ is measured at room temperature using a plate reader (MTP-32 manufactured by Corona Electric Co., Ltd.) with an absorbance of 450 nm (control wavelength 630 nm). Further, the binding amount is obtained from a calibration curve prepared beforehand with bovine serum albumin.

  Since the implant of the present invention is less prone to inflammation when implanted in a living body, it can be used as an implant that is embedded in various living bodies. From the viewpoint of suppressing inflammation, an implant that is preferably a medical device for implanting a living body. It can be used for bone fixing agents, bone cages, catheters, guide wires, stents, artificial blood vessels and artificial joints.

The method for producing an implant of the present invention is at least one selected from the group consisting of spraying, dipping and coating a solution (B1) having a concentration of 5 to 100 μg / ml of the implant coating agent on the surface of the implant material (C1). It is a manufacturing method of an implant including the process of coat | covering the surface of an implant material (C1) with a seed | species method.
Implant material (C1): Implant material made of a polymer material and / or a ceramic material.

  The concentration of the coating agent for implants in the solution (B1) is preferably 5 to 50 μg / ml from the viewpoint of suppressing inflammation.

  The solvent of the solution (B1) is not limited as long as it can dissolve 5 μg / ml or more of the implant coating agent. For example, water, methanol, ethanol, dimethyl sulfoxide, etc. are mentioned. From the viewpoint of the solubility of (A), water is preferred.

In the implant material (C1), examples of the polymer material include MPC (2-methacryloyloxyethyl phosphorylcholine) polymer, PVA (polyvinyl alcohol), polystyrene, polyethylene, and polypropylene.
Examples of the ceramic material include alumina, zirconia, anatase, rutile, and hydroxyapatite.
The shape of the implant material (C1) is not particularly limited. For example, the shape of an artificial hip joint, the shape of an artificial knee joint, the shape of an artificial tooth root, the shape of a bone prosthesis / material, etc. No. 14), published by Iwanami Shoten Co., Ltd. ”.

  In the production method of the present invention, the solution (B1) containing the implant coating agent is applied to the surface of the implant material (C1) by at least one method selected from the group consisting of spraying, dipping and coating. By making it exist on the surface, the coating agent for implant in the solution (B1) is chemically bonded {covalent bond, ionic bond and / or hydrogen bond, etc.} or physical adsorption {adsorption by van der Waals force, etc. to the implant material (C1). } To coat the surface of the implant material.

The method for producing an implant of the present invention includes the step of applying a voltage between the implant material (C2) and the electrode in the solution (B2) in which the concentration of the implant coating agent is 5 to 100 μg / ml. Is the method.
Implant material (C2): Implant material made of a metal material and / or an ion-bonding ceramic material.

  The concentration of the coating agent for implants in the solution (B2) is preferably 5 to 50 μg / ml from the viewpoint of suppressing inflammation.

The solvent of the solution (B2) is not limited as long as it can dissolve 5 μg / ml or more of the implant coating agent. Examples include water, methanol, ethanol, and dimethyl sulfoxide. From the viewpoint of solubility, water is preferred.
It is preferable to dissolve the inorganic electrolyte in the solvent. As the inorganic electrolyte to be dissolved, chlorides of alkali metals and alkaline earth metals can be used, and specific examples include sodium chloride, potassium chloride, and calcium chloride. By dissolving the inorganic electrolyte, the solution has electrical conductivity, and the implant coating agent can move toward the implant material (C2). The concentration of the inorganic electrolyte is preferably 1 to 5% by weight and more preferably 2 to 4% by weight with respect to the weight of the solution (B2). When the concentration of the inorganic electrolyte is 1% by weight or more, the electrical conductivity of (B2) is preferably improved. A concentration of the inorganic electrolyte of 5% by weight or less is preferable because ions in the inorganic electrolyte can be prevented from adsorbing to the implant material surface.

In the implant material (C2), examples of the metal material include titanium, stainless steel, zirconium, tantalum, platinum, gold, and vitalium, and composite materials composed of two or more of these (transplant and artificial organ, Iwanami course) Modern medical basics 14, published by Iwanami Shoten Co., Ltd. and Japanese Patent Laid-Open No. 7-88174). Examples of the composite material include titanium alloys (such as Ti-6Al-4V and Ti-6Al-2Nb-1Ta) and cobalt chromium alloys.
Specific examples of the ion binding ceramic include alumina, zirconia, anatase, rutile, and hydroxyapatite. (C2) is preferably titanium or a titanium alloy from the viewpoint of biocompatibility.
The shape of the implant material (C2) is not particularly limited. For example, the shape of an artificial hip joint, the shape of an artificial knee joint, the shape of an artificial tooth root, the shape of a bone prosthesis / material, etc. No. 14), published by Iwanami Shoten Co., Ltd. ”.

In the production method of the present invention, a voltage is applied between the implant material (C2) and the electrode in the solution (B2) in which the concentration of the implant coating agent is 5 to 100 μg / ml, whereby the implant coating agent is implanted into the implant. It can be fixed to the surface of the material (C2) by an electrochemical reaction.
An electrochemical reaction is a reaction in which the electrochemical potential changes in an electrochemical system due to other dynamic factors, such as movement of a substance toward an electrode surface, adsorption to the electrode surface, dissociation at the electrode surface, and transfer of electrons. It is a reaction that goes through the process of.
Protons are added to the side chain amino groups such as proline (P) in the implant coating agent to form quaternary ammonium cations and move toward the cathode. Quaternary ammonium cations are adsorbed on the cathode surface and dissociated into amino groups and protons on the cathode surface. Electrons are given to protons from the cathode, and hydrogen gas is generated. Since the electrons of the lone pair of amino group are shared with the free electrons of the metal that is the cathode, a strong bond is formed between the amino group of the implant coating agent and the implant coating agent that is the cathode. This strong bond is retained after stopping.

  As the method for producing the implant of the present invention, for example, the implant material (C2) and the electrode are immersed in the solution (B2), the implant material (C2) is used as a cathode, the electrode is used as an anode, and a voltage is applied between both electrodes. Thus, a method of fixing the implant coating agent to the surface of the implant material (C2) by an electrochemical reaction may be mentioned.

In the production method of the present invention, the voltage applied between the cathode and the anode is preferably 0.1 to 10V, and more preferably 1 to 7V. When the voltage is 0.1 V or more, the time required for forming a coating by fixing the implant coating agent to the cathode surface can be shortened. It is preferable that the electrical voltage is 10 V or less because the generation of bubbles on the metal surface can be suppressed and a uniform film is formed.
The current density with respect to the surface area of the cathode is preferably 5 × 10 ^{−8 to} 5 × 10 ⁻⁵ A / dm ² , more preferably 1 × 10 ⁻⁷ to 1 × 10 ⁻⁵ A / dm ² . When the current density is 5 × 10 ⁻⁸ A / dm ² or more, the time required for forming the coating by fixing the implant coating agent to the cathode surface can be shortened. It is preferable for the current density to be 5 × 10 ⁻⁵ A / dm ² or less because the generation of bubbles on the metal surface can be suppressed and a uniform film can be formed.

In the present invention, when the implant coating agent is fixed to the surface of the implant material (C1) or (C2), the covering area representing the area (N) for fixing the implant coating agent to the surface area (M) that adheres to the living tissue. The rate ((N) / (M)) is preferably 50 to 100%, particularly preferably 80 to 100%, from the viewpoint of suppressing inflammation.
The binding amount, inflammation suppression point of view, is preferably 1-1000 ng / mm ^2, particularly preferably from 5~1000ng / mm ^2.

The binding amount of the implant coating agent in the implant obtained by the production method of the present invention is a value measured by the following measurement method.
<Measurement of binding amount>
A mixed solution (C) of Reagent A solution: Reagent B solution: Reagent C solution = 25: 24: 1 attached to Micro BCA ^™ protein assay kit (manufactured by THERMO Fisher Scientific) is prepared. Add the mixture (C) at a rate of 312.5 μL / cm ² to the surface area of the part to be measured for the binding amount of the coating agent for the implant in the implant, and evenly adhere to the surface of the part to be measured at 37 ° C. for 2 hours. Leave still.
After 2 hours of standing, the chelate production amount of bicinchoninic acid (BCA) and Cu ⁺ is measured at room temperature using a plate reader (MTP-32 manufactured by Corona Electric Co., Ltd.) with an absorbance of 450 nm (control wavelength 630 nm). Further, the binding amount is obtained from a calibration curve prepared beforehand with bovine serum albumin.

  Since the implant obtained by the production method of the present invention is less prone to inflammation when implanted in a living body, it can be used as an implant that is implanted in various living bodies. It can be used for implants, bone fixing agents, bone cages, catheters, guide wires, stents, artificial blood vessels and artificial joints.

  Hereinafter, although an example and a comparative example explain the present invention further, the present invention is not limited to these. Hereinafter, unless otherwise specified, “%” represents “% by weight” and “parts” represents “parts by weight”.

<Example 1>
○ Manufacture of artificial protein (A1) coated polystyrene film (PS1) GAGAGS produced by genetically modified Escherichia coli and purified by column chromatography according to the method described in Examples in JP-T-3-502935 (GAGAGS) in which 4 sequences (13) are continuous In 12 polypeptide chains (S1-1) in ₄ sequences (14) and in polypeptide chains (Y1-1) in which 8 GVGVP sequences (1) are continuous It has 13 (GVGVP) ₄ GKGVP (GVGVP) ₃ sequences (19) (Y'1-1) in which one of V (valine) is replaced by K (lysine), and these are chemically bonded alternately. A molecular substance having a structure in which one polypeptide chain (S1-2) of (GAGAGS) ₂ sequence (15) in which two GAGAGS sequences (13) are consecutively bonded is chemically bonded. An artificial protein (SELP8K) (A1) having a sequence (21) of about 70 kDa was obtained. This artificial protein (A1) was used as a coating agent for implants.
The artificial protein (A1) was dissolved in deionized water at a concentration of 10 μg / mL to prepare an artificial protein (A1) solution. A polystyrene film (manufactured by BIO-BIK, product name “balance dish”, 0.5 cm × 0.5 cm) is completely immersed in the artificial protein (A1) solution, and the solution is ₂ under conditions of 37 ° C. and 5% by volume CO 2. Let stand for hours.
After standing for 2 hours, it was immersed in deionized water three times to obtain a polystyrene film (PS1) coated with an artificial protein (A1). The covering area ratio was 100%.

<Example 2>
Preparation of artificial protein (A2) -coated polystyrene film (PS2) In the same manner as in Example 1, a polypeptide chain (S1-2) having two GAGAGS sequences (13) (GAGAGS) ₂ sequence (15) and (GVGVP) ₄ GKGVP (GVGVP) _{3 The} sequence (19) has 17 polypeptide chains (Y′1-1), each having a structure in which these are alternately chemically bonded, and a molecular mass of about 77 kDa An artificial protein (SELP0K) (A2) of (22) was prepared. This artificial protein (A2) was used as an implant coating agent.
The artificial protein (A2) was dissolved in deionized water at a concentration of 10 μg / mL to prepare an artificial protein (A2) solution. A polystyrene film (0.5 cm × 0.5 cm) was completely immersed in the artificial protein (A2) solution and allowed to stand at 37 ° C. under 5% by volume CO ₂ for 2 hours.
After standing for 2 hours, it was immersed three times in deionized water to obtain a polystyrene film (PS2) coated with an artificial protein (A2). The covering area ratio was 100%.

<Example 3>
Preparation of artificial protein (A3) -coated polystyrene film (PS3) In the same manner as in Example 1, a polypeptide chain (S1-2) having two GAGAGS sequences (13) (GAGAGS) ₂ sequence (15) was obtained. (GVGVP) ₄ GKGVP (GVGVP) ₁₁ sequence in which one of V (valine) in 16 and 16 GVGVP sequences (1) in a polypeptide chain (Y11) was replaced with K (lysine) 20) A sequence having a molecular mass of about 71 kDa (23) having 8 (Y′1-2) and chemically bonding with {(S1-2) (Y′12) (S1-2)} ₁₆ ) Artificial protein (SELP415K) (A3). This artificial protein (A3) was used as a coating agent for implants.
The artificial protein (A3) was dissolved in deionized water at a concentration of 10 μg / mL to prepare an artificial protein (A3) solution. A polystyrene film (0.5 cm × 0.5 cm) was completely immersed in the artificial protein (A3) solution and allowed to stand at 37 ° C. under 5% by volume CO ₂ for 2 hours.
After standing for 2 hours, it was immersed in deionized water three times to obtain a polystyrene film (PS3) coated with an artificial protein (A3). The covering area ratio was 100%.

<Example 4>
Preparation of artificial protein (A4) -coated polystyrene film (PS4) In the same manner as in Example 1, a polypeptide chain (S1-2) having two GAGAGS sequences (13) (GAGAGS) ₂ sequence (15) was obtained. (GVGVP) ₄ GKGVP (GVGVP) ₁₁ sequence in which one of V (valine) in 16 polypeptide chains (Y11) in which 16 GVGVP sequences (1) are continuous is substituted with K (lysine) ) 6 (Y′12) and ₄ polypeptide chains (S1-1) of (GAGAGS) ₄ sequence (14) with ₄ consecutive GAGAGS sequences (13), which are chemically bonded alternately Thus, an artificial protein (SELP815K) (A4) having a sequence (24) having a molecular mass of about 65 kDa was prepared. This artificial protein (A4) was used as a coating agent for implants.
The artificial protein (A4) was dissolved in deionized water at a concentration of 10 μg / mL to prepare an artificial protein (A4) solution. A polystyrene film (0.5 cm × 0.5 cm) was completely immersed in the artificial protein (A4) solution and allowed to stand at 37 ° C. under 5% by volume CO ₂ for 2 hours.
After leaving still for 2 hours, it was immersed in deionized water three times to obtain a polystyrene film (PS4) coated with an artificial protein (A4). The covering area ratio was 100%.

<Comparative Example 1>
A polystyrene film (0.5 cm × 0.5 cm) was immersed in deionized water and allowed to stand for 2 hours at 37 ° C. and 5% by volume CO ₂ .
After standing for 2 hours, it was immersed in deionized water three times to obtain a comparative polystyrene film (PS5) that was not coated with an artificial protein.

<Example 5>
○ Manufacture of artificial protein (A1) coated titanium plate (PT1) The artificial protein (A1) was dissolved in a 0.9% by weight sodium chloride aqueous solution so as to have a concentration of 5 μg / ml, and had a height of 200 mm and a bottom diameter of 135 mm Placed in a glass electrolytic cell. The titanium plate is completely immersed in the liquid with the platinum electrode as the anode and the titanium plate (0.5 cm × 0.5 cm × 0.1 mm) (product name “Titanium / plate” manufactured by Nilaco Corporation) as the cathode. In this way, while stirring the liquid by rotating the stirrer with a magnetic stirrer, a voltage of 3.0 V was applied between the two electrodes, and electricity was applied for 10 minutes to perform an electrochemical reaction. After stopping energization, it was washed three times in 5 ml of deionized water and immediately removed, and then dried for 2 hours in a 60 ° C smooth air dryer, and the titanium surface was coated with artificial protein (A1). A plate (PT1) was obtained. The covering area ratio was 100%.

<Example 6>
Preparation of artificial protein (A2) -coated titanium plate (PT2) A titanium plate (PT2) was obtained in the same manner as in Example 5 except that the artificial protein (A1) was changed to the artificial protein (A2). The covering area ratio was 100%.

<Example 7>
Preparation of artificial protein (A3) coated titanium plate (PT3) A titanium plate (PT3) was obtained in the same manner as in Example 5 except that the artificial protein (A1) was changed to the artificial protein (A3). The covering area ratio was 100%.

<Example 8>
Preparation of artificial protein (A4) coated titanium plate (PT4) A titanium plate (PT4) was obtained in the same manner as in Example 5 except that the artificial protein (A1) was changed to the artificial protein (A4). The covering area ratio was 100%.

<Comparative example 2>
A comparative titanium plate (PT5) was obtained in the same manner as in Example 5 except that the artificial protein (A1) was not used and only a 0.9 wt% sodium chloride aqueous solution was used.

The binding amount of the coating agent for implants in the polystyrene films (PS1) to (PS5) and the titanium plates (PT1) to (PT5) was determined by the following measurement.
<Measurement of binding amount>
A mixed solution (C) of Reagent A solution: Reagent B solution: Reagent C solution = 25: 24: 1 attached to Micro BCA ^™ protein assay kit (manufactured by THERMO Fisher Scientific) was prepared. 156.3 μL of each of the polystyrene films (PS1) to (PS5) and 218.8 μL of the mixed liquid (C) to each of the titanium plates (PT1) to (PT5) were evenly adhered to the surface at 37 ° C. Let stand for 2 hours.
Two hours after standing, the amount of chelate produced between bicinchoninic acid (BCA) and Cu ⁺ was measured at room temperature using a plate reader (MTP-32 manufactured by Corona Electric Co., Ltd.) with an absorbance of 450 nm (control wavelength 630 nm). Furthermore, the binding amount was obtained from a calibration curve prepared beforehand with bovine serum albumin.

<Evaluation: Measurement of inflammatory reaction>
Polystyrene films (PS1) to (PS5) and titanium plates (PT1) to (PT5) were implanted in the rat back skin, respectively. Three test specimens were prepared for each kind of film or plate. The occurrence of inflammation was observed for each specimen. As observation items, bleeding, enveloping, new bone formation, presence / absence of discoloration and the extent (spreading, thickness, etc.) in the tissues surrounding the test sample were observed with the naked eye. The results are shown in Tables 1 and 2. In the table, with respect to the inflammatory reaction, a certain thing is represented by + and a non-significant thing is represented by-, and each result of three test bodies is shown in parentheses. In addition, the overall evaluation of two or more specimens having an inflammatory reaction was rated as +. Moreover, when it corresponded to at least 1 item of an observation item, it was judged that there was inflammation. Furthermore, as for the result of visual observation, the item in which symptoms were observed was described for at least one test body.

  From the results of Tables 1 and 2, it can be seen that inflammation can be reduced by coating the implant material with the coating agent for implants of the present invention. Moreover, even if inflammation occurs, it turns out that inflammation heals early.

  Since the coating agent for implants of the present invention can reduce inflammation caused by implants, it is not only a coating agent for coating implant materials {metal materials, polymer materials, ceramics, etc.) used in medical and dentistry, but also contacts. It is also useful as a coating agent for lenses. In addition, according to the method for producing an implant of the present invention, an implant with less inflammation due to the implant material can be produced, and the produced implant is useful as an implant used in medicine and dentistry.

Claims (7)

An implant coating agent comprising an artificial protein (A),
(A) has a polypeptide chain (Y) having 2 to 200 consecutive GVGVP sequences (1 ) as the amino acid sequence (X) and / or the following polypeptide chain (Y ′), Y) and (Y ') total number of is 1 to 100, (hydrophobic degree 0.2-1.2 der of a) Ri, (a) is (GAGAGS) ₄ sequence (14) and and / or (GAGAGS) ₂ sequence _{(15), (GVGVP) 4} GKGVP (GVGVP) 3 sequence (19) or (GVGVP) ₄ GKGVP (GVGVP) ₁₁ sequence (20) and a Ru der engineered proteins implants having Coating agent.
Polypeptide chain (Y ′): A polypeptide chain in which 0.1 to 10% of the number of amino acids in (Y) is substituted with lysine and / or arginine. The artificial protein (A) has a GAGAGS sequence (13), and the number of the GAGAGS sequence (13) and the total number of the amino acid sequence (X) and the following amino acid sequence (X ′) in one molecule of the artificial protein (A) The implant coating agent according to claim 1, wherein the ratio (sequence (13): amino acid sequence (X) and (X ′)) is 1: 2 to 1:20.
Amino acid sequence (X ′): GKGVP sequence in which one amino acid in the GVGVP sequence (1) is replaced with lysine . The implant coating agent according to claim 1 or 2 , wherein the artificial protein (A) has a molecular weight of 15 to 200 kDa as determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis). The artificial protein (A) is (GAGAGS) ₄ sequence (14) And (GVGVP) ₄ GKGVP (GVGVP) ₃ sequence (19) (GAGAGS) ₂ sequence (15) and (GVGVP) ₄ GKGVP (GVGVP) ₃ artificial protein having sequence (19), (GAGAGS) ₂ sequence (15) and (GVGVP) ₄ GKGVP (GVGVP) ₁₁ The implant coating agent according to any one of claims 1 to 3 , which is an artificial protein having the sequence (20) . An implant having a surface coated with the coating agent for implants according to any one of claims 1 to 4 . At least selected from the group consisting of spraying, dipping and coating a solution (B1) having a concentration of the coating agent for implants according to any one of claims 1 to 4 of 5 to 100 µg / ml on the surface of the implant material (C1). The manufacturing method of an implant including the process of coat | covering the surface of an implant material (C1) by 1 type of methods.
Implant material (C1): Implant material made of a polymer material and / or a ceramic material. An implant comprising: a step of applying a voltage between an implant material (C2) and an electrode in a solution (B2) having a concentration of the implant coating agent according to any one of claims 1 to 4 of 5 to 100 µg / ml. Production method.
Implant material (C2): Implant material made of a metal material and / or an ion-bonding ceramic material.