JPH07267876A - Composition for stabilizing cell growth factor - Google Patents

Abstract

PURPOSE:To obtain a composition, capable of stably retaining the activities of a cell growth factor for a long period and useful as a regenerating agent for a periodontal tissue, a wound curing agent, etc., by blending the cell growth factor with water-soluble polysaccharides, etc. CONSTITUTION:This composition is obtained by blending (A) a cell growth factor such as a cell growth factor derived from a blood platelet with (B) water- soluble polysaccharides such as an alginate or xanthan gum or a water-soluble vinyl polymer such as a carboxyvinyl polymer and preferably further (C) one or two or more selected from citric acid (salts), laurylsulfuric acid (salts) and polysorbates.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the stabilization of cell growth factors. More specifically, the present invention relates to a technique for stably incorporating a platelet-derived cell growth factor into a gel base. The present invention relates to a technique for promoting stabilization of a composition containing a cell growth factor such as a periodontal tissue regenerating agent and a wound healing agent.

[0002]

BACKGROUND OF THE INVENTION Platelet-derived cell growth factor (PDGF) is a polypeptide with a molecular weight of about 30,000 released from platelets, which is often present at the wound site. It was discovered as a factor that promotes basic functions such as proliferation (Antoniad
es: Proc. Natl. Acad. Sci. USA, 78,7314-7317,198
1). After that, it became clear that PDGF is released not only from platelets but also from monocytes, epithelial cells, muscle cells, etc. (Ross et al: Cell, 45,155-169,1986), and the growth and tissue repair of individuals, It has been shown to play a crucial role in regeneration (Ross: Lancet, May, 27,1179-1182,
1989). Further surgical administration of PDGF resulted in cutaneous wounds (Lynch et al: J. Clin. Invest., 84,640-646,198.
9), ulcer (Pierce et al: J. Cell Biochem., 45,319-3
26, 1991) and also promote healing of destroyed tissue in periodontal disease (USP5124316). As described above, PDGF is expected to be applied as an external preparation for promoting wound healing, but since a formulation that stably contains PDGF for a long period of time and retains physiological activity has not been established, it has not yet been commercialized. Absent. Although water-soluble polysaccharides (EP267015) have been shown in the formulation of cell growth factor as a general topical agent, since individual cell growth factors have different chemical and physical properties, PD
There were still points to be improved in order to stably contain GF. In addition, application of collagen (EP243719) as a carrier has been proposed, but the problem of harmful effects on the living body due to antigenicity has not been solved.

[0003]

Means for Solving the Problems The present invention has found that the activity of PDGF is stably maintained by mixing PDGF with sodium alginate, xanthan gum or carboxyvinyl polymer, and the PDGF-containing compound obtained by combining them is obtained. The external gel preparation is completed and provided as a stable PDGF external preparation. P
DGF consists of two kinds of monomers called α chain and β chain, and three kinds of PDGF-AB heterodimer, -AA homodimer, and -BB homodimer exist in nature depending on the combination, but in the present invention. Thus, both types of PDGF are stabilized. PDGF can be purified using human platelets as a material, or can be obtained by a gene recombination method. PDGF purified by these methods is available from GIBCO, Inc., Upst, USA
It is commercially available from ate Biotechnology, Inc. and can be easily obtained. Although the concentration of PDGF contained in the formulation depends on the application, the present invention can stabilize PDGF activity at any concentration.

Sodium alginate, xanthan gum,
Carboxyvinyl polymers are listed in the standard for pharmaceutical additives, and they are often used as water-soluble bases.
There is no problem in safety. In order to obtain a gel for external use using these as materials, in all cases, the weight percentage is 0.0.
By including 5-5%, preferably 1-2%,
The desired stabilizing formulation can be obtained.

Further, pharmacologically acceptable additives can be added depending on the use. Among these additives, especially when citric acid and its salt, sodium lauryl sulfate and polysorbate 60, 80 are added,
This is preferable because the stability of GF is further improved. The compounding amount of these additives is 0.001 with respect to the composition.
Is about 5%, preferably about 0.08 to 2%.

[0006]

[Examples] Examples as agents for periodontal tissue regeneration are described below. Furthermore, experimental examples showing that PDGF is stably contained in these formulations and the activity is retained, and examples of use as agents for periodontal tissue regeneration are shown. The present invention is not limited to these examples. In this example, the required amount of each component was weighed based on the following examples, purified water was added and the mixture was stirred to produce an ointment.

Example 1 Ingredient content (weight) Platelet-derived cell growth factor 1 mg Xanthan gum 0.5 g Purified water Appropriate amount Total amount 100 g

Example 2 Component content (weight) Platelet-derived cell growth factor 1 mg Xanthan gum 1 g Purified water Appropriate amount Total 100 g

Example 3 Component content (weight) Platelet-derived cell growth factor 1 mg Xanthan gum 5 g Purified water Appropriate amount Total 100 g

Example 4 Ingredient content (weight) Platelet-derived cell growth factor 1 mg Xanthan gum 1 g Citric acid 80 mg Purified water Appropriate amount Total amount 100 g

Example 5 Component content (weight) Platelet-derived cell growth factor 1 mg Xanthan gum 1 g Polysorbate 60 1 g Purified water Appropriate amount Total amount 100 g

Example 6 Component content (weight) Platelet-derived cell growth factor 1 mg Xanthan gum 1 g Polysorbate 80 1 g Purified water Appropriate amount Total amount 100 g

Example 7 Component content (weight) Platelet-derived cell growth factor 1 mg Xanthan gum 1 g Sodium lauryl sulfate 1 g Purified water Appropriate amount 100 g

Example 8 Component content (weight) Platelet-derived cell growth factor 1 mg Sodium alginate 0.5 g Purified water Appropriate amount Total amount 100 g

Example 9 Component content (weight) Platelet-derived cell growth factor 1 mg Sodium alginate 1 g Purified water Appropriate amount Total amount 100 g

Example 10 Component content (weight) Platelet-derived cell growth factor 1 mg Sodium alginate 5 g Purified water Appropriate amount Total amount 100 g

Example 11 Component content (weight) Platelet-derived cell growth factor 1 mg Sodium alginate 1 g Citric acid 80 mg Purified water Appropriate amount Total amount 100 g

Example 12 Component content (weight) Platelet-derived cell growth factor 1 mg Sodium alginate 1 g Polysorbate 60 1 g Purified water Appropriate amount Total amount 100 g

Example 13 Component content (weight) Platelet-derived cell growth factor 1 mg Sodium alginate 1 g Polysorbate 80 1 g Purified water Appropriate amount Total amount 100 g

Example 14 Composition (weight) Platelet-derived cell growth factor 1 mg Sodium alginate 1 g Sodium lauryl sulfate 1 g Purified water Appropriate amount Total amount 100 g

Example 15 Component content (weight) Platelet-derived cell growth factor 1 mg Carboxyvinyl polymer 0.5 g Purified water Appropriate amount Total 100 g

Example 16 Component content (weight) Platelet-derived cell growth factor 1 mg Carboxyvinyl polymer 1 g Purified water Appropriate amount Total 100 g

Example 17 Component content (weight) Platelet-derived cell growth factor 1 mg Carboxyvinyl polymer 5 g Purified water Appropriate amount Total 100 g

Example 18 Component content (weight) Platelet-derived cell growth factor 1 mg Carboxyvinyl polymer 1 g Citric acid 80 mg Purified water Appropriate amount Total 100 g

Example 19 Component content (weight) Platelet-derived cell growth factor 1 mg Carboxyvinyl polymer 1 g Polysorbate 60 1 g Purified water Appropriate total amount 100 g

Example 20 Ingredient content (weight) Platelet-derived cell growth factor 1 mg Carboxyvinyl polymer 1 g Polysorbate 80 1 g Purified water Appropriate amount Total amount 100 g

Example 21 Ingredient content (weight) Platelet-derived cell growth factor 1 mg Carboxyvinyl polymer 1 g Sodium lauryl sulfate 1 g Purified water Appropriate amount Total amount 100 g

Stability test of PDGF Experiment 1: Experiment for demonstrating that PDGF is stably retained as a substance in three kinds of bases Method Example 1, 2, 3 (xanthan gum), Example 8, 9 10 (sodium alginate), Example 15,
16 and 17 (carboxyvinyl polymer), and for comparison, a mixture containing 1% of carboxymethylcellulose or polyvinylpyrrolidone,
Each 8 ml of silicon-coated tube (manufactured by Sigma) was dispensed, and 4 tubes of each were stored at 4 ° C and 40 ° C.
Immediately after storage and after 1, 2 and 4 months, the tubes containing each sample were taken out from the storage at 4 ° C. and 40 ° C. one by one, and the amount of PDGF in the solution containing 0.9 ml of water was measured. PDG labeled with 125 Iodine for measurement
A radioimmunoassay method (manufactured by Amersham) using anti-rh-PDGF antibody with F as a tracer was used.
The PDGF residual rate in each sample was calculated as a relative ratio to the PDGF amount (0.05 mg / sample) included in the calculation at the time of sample preparation. The results are shown in Table 1.

[0029]

[Table 1]

As shown in the above table, xanthan gum, sodium alginate, and carboxyvinyl polymer can stably retain PDGF for a long period of time as compared with carboxymethylcellulose, polyvinylpyrrolidone, and an aqueous solution.

Experiment 2: Experimental procedure demonstrating that PDGF is retained as a substance more stably in the base and the additives Method Examples 4, 5, 6, 7 (xanthan gum and additives), Example 11, 12, 13, 14 (sodium alginate and additives) and the mixture shown in Examples 18, 19, 20, 21 (carboxyvinyl polymer and additives).
0.1 ml each was dispensed into 8 silicon-coated tubes (manufactured by Sigma), and 4 tubes each were stored at 4 ° C. and 40 ° C., and the PDGF amount (relative ratio) was calculated by the same method as in Experiment 1. . The results are shown in Table 2.

[0032]

[Table 2]

As shown in the table above, by adding various additives, ie, polysorbate 60, polysorbate 80, sodium lauryl sulfate, and citric acid, to xanthan gum, sodium alginate, and carboxyvinyl polymer, PDGF can be more stably and long-term. Can be held.

Experiment 3: Experiment showing that the activity of PDGF is stably maintained Method Example 4, 5, 6, 7 (xanthan gum and additive), Examples 11, 12, 13, 14 (with sodium alginate) Additive), the mixture shown in Examples 18, 19, 20, 21 (carboxyvinyl polymer and additive), and a mixture containing 1% of carboxymethylcellulose or polyvinylpyrrolidone for comparison, in 8 ml portions of 0.1 ml each. It was dispensed into a silicon-coated tube (manufactured by Sigma) and stored at 4 ° C and 40 ° C, 4 tubes each. Immediately after storage, and after 1, 2, and 4 months, remove the tubes containing each sample from the storage at 4 ° C and 40 ° C, 1% each, and 1%
Dulbecco's modified Eagle M containing fetal bovine serum (FBS)
The rh-PDGF activity in a solution containing 0.9 ml of EM (DMEM) medium (manufactured by Gibco) was measured. For the activity measurement, DNA by incorporation of thymidine labeled with tritium in mouse Balb / c3T3 cells was used.
A synthetic assay was used. Separately, as a PDGF standard sample, 1 mg / ml PDGF was dispensed 0.1 ml each into a silicon-coated tube and stored at -20 ° C.
0.9 ml of DMEM medium containing BS was added, and PDGF activity was measured by the same method. PD in each sample
GF activity was calculated as the relative ratio of this standard sample to PDGF activity. The results are shown in Table 3.

[0035]

[Table 3]

As shown in the above table, the activity of PDGF was stabilized for a long period of time by adding various additives such as polysorbate 60, polysorbate 80, sodium lauryl sulfate and citric acid to xanthan gum, sodium alginate and carboxyvinyl polymer. Can be held at.

Experiment 4: Action on Periodontal Tissue Regeneration Process after Canine Gingival Detachment Curing Surgery Method The action of various PDGF formulations on the periodontal tissue regenerating process after canine gingival ablation curettage surgery was examined by a pathological quantitative method. The upper and lower premolars, in which a healthy periodontal tissue was established by brushing, etc., were subjected to gingival detachment curettage surgery according to a conventional method. At this time, a reference point called a notch was added to the root surface before and after the removal of the alveolar bone in order to use it as a reference point for later histopathological quantification. The specimens were the same gel agents as in Examples 2, 9, 16 shown in the Examples and Examples 4, 12, 13, 21 further containing a stabilizer and stored in a silicon-coated tube at 4 ° C. for 2 months. As a control, the right upper and lower jaws were administered immediately after compounding the drug with the same formulation. After surgery, the gingival flap is repositioned,
Suture and pack protection was applied for 1 week. Evaluation is 4 after surgery
After the test site was collected in the week and a tissue sample was prepared by a conventional method, the distance between the sites was measured under a microscope using an eyepiece micrometer and quantified according to the following criteria. Indicated.

[0038]

[Formula 1]

[0039]

[Formula 2]

[0040]

[Table 4]

As shown in the above table, xanthan gum, sodium alginate, and carboxyvinyl polymer exhibited the same activity as that immediately after the formulation in suppressing downgrowth of epithelium and promoting fibrotic adhesion after storage for 2 months. As is clear from the above results, the formulation based on xanthan gum, sodium alginate, and carboxyvinyl polymer is useful for maintaining the tissue regeneration activity of PDGF.

Experiment 5: Effect of rat dorsal wound on tissue regeneration process Method The test was carried out using 3 Wistar rat rats per group. Under ether anesthesia, the hair on the back was clipped with a clipper, and the back skin was disinfected with 70% ethanol. The position of 15 mm was punched out with a chisel for woodworking with a blade length of 15 mm, and two scratches were created at the same time. Immediately after suturing the central part of the wound part, a gel similar to that shown in Examples 2, 9, 16 and further Examples 4, 12, 13, and 21 containing a stabilizer was prepared immediately after preparation and silicone. Each wound was stored at 4 ° C. for 2 months in a coated tube and applied at about 500 mg per wound once a day for 5 days.
Thread removal on the 4th day, slaughter on the 6th day, peeling back skin,
The wound was cut into a strip shape with a width of about 8 mm using a scalpel. Using a rheometer, the tension required for dehiscence of wound (wound resistance) was measured. The results are shown in Table 5.

[0043]

[Table 5]

As shown in the above table, xanthan gum, sodium alginate, and carboxyvinyl polymer showed the same tensile strength against wound strength immediately after preparation even after storage for 2 months. As is clear from the above results, xanthan gum,
A formulation based on sodium alginate or carboxyvinyl polymer is useful for maintaining the activity of PDGF.

[0045]

INDUSTRIAL APPLICABILITY According to the present invention, the activity of the cell growth factor can be stably maintained for a long period of time by blending the cell growth factor with a water-soluble polysaccharide or a water-soluble vinyl polymer. The cell growth factor is preferably platelet-derived cell growth factor, the water-soluble polysaccharide is preferably sodium alginate or xanthan gum, and the water-soluble vinyl polymer is preferably carbovinyl polymer.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location A61K 9/00 V 47/12 J 47/20 J 47/32 J 47/34 J 47/36 J // C07K 14/475 8318-4H (A61K 47/32 47:12) (A61K 47/32 47:34) (A61K 47/32 47:20) (A61K 47/36 47:12) (A61K 47/36 47:34) (A61K 47/36 47:20) A61K 37/24 ADA

Claims (4)

[Claims] 1. A composition containing a cell growth factor and a water-soluble polysaccharide or a water-soluble vinyl polymer. 2. The composition according to claim 1, wherein the cell growth factor is platelet-derived cell growth factor. 3. The composition according to claim 1, wherein the polysaccharide polymer substance is alginate or xanthan gum, and the water-soluble vinyl polymer is carboxyvinyl polymer. 4. The composition according to claim 1, further comprising one or more selected from citric acid and salts thereof, lauryl sulfate and salts thereof, and polysorbate.