JP2915907B2 - Insulin binding carrier - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an insulin-binding carrier comprising a protein or polypeptide having an insulin-binding activity as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, insulin receptors present on cell surfaces are known as proteins and polypeptides that bind insulin. In addition, regarding the non-receptor protein, the present inventors have previously described fibronectin, laminin, chondronectin, fibrinogen,
It has been found that vitronectin and fibrin have an insulin-binding ability, and they have been provided as a composition for an insulin-binding carrier (JP-A-1-85934). On the other hand, as for insulin-like growth factor (IGF-I: somatomedin C) and IGF-II, which are very similar in structure to insulin, IGF-binding protein, which is a non-receptor binding protein, has been extracted from blood for a long time. It has been isolated and purified. That is, unlike insulin, IGF forms a polymer complex with a binding protein in blood and exists in a large number in a biologically inactive state. By forming a complex, the half-life in vivo is increased, stabilized, and involved in IGF homeostasis in vivo.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have further studied a novel insulin-binding carrier. That is, an object of the present invention is to provide a novel insulin binding carrier that can be used as an insulin carrier.

[0004]

SUMMARY OF THE INVENTION In general, the present invention comprises collagen type I, type II, type III, type IV, type V, and insulin binding sites each having insulin binding activity, The present invention relates to a carrier for insulin binding, comprising as an active ingredient at least one protein or polypeptide selected from the group consisting of collagen V-type degrading polypeptides having insulin binding activity.

[0005] The present inventors have thought that there may be a protein having an insulin binding activity or a protein which is a carrier, and have made intensive studies. Such a protein or polypeptide can regulate the homeostasis of insulin having a growth factor activity and a hormonal activity, and when administering insulin to various diseases represented by diabetic patients, the insulin or insulin can be regulated. Build safe, non-immunogenic drugs with long-term targeting and long-lasting action, maximize insulin effects, and build new formulations and drug delivery systems with no side effects It becomes possible. For example, a transdermal drug that provides a growth-promoting reagent that is effective for biochemical research, provides useful cosmetics that increase the proliferation of epidermal cells, and prevents skin aging, and that promotes wound healing after trauma or surgery And a therapeutic agent are provided, and an insulin preparation which is used for the treatment of diabetes and has high durability and no side effects is provided. The present inventors conducted research to solve the above-mentioned problems, and confirmed that collagens I, II, III, IV, and V, which are biological components, have insulin-binding ability, and that collagen V has a specific insulin-binding ability. It was found to have an active site.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. Collagen is a main protein constituting connective tissue, and three α chains form a right-handed helical structure to constitute a collagen molecule. The structure of the α chain differs somewhat depending on the tissue, and collagen is classified into types I, II, III, IV, V
Classified into types. Insulin is a peptide hormone consisting of A and B chains, and is known to exhibit various physiological functions.

[0007] Peroxidase-labeled insulin is used to search for proteins having insulin binding activity. That is, the insulin binding activity can be detected by binding the insulin to a protein sample and measuring the peroxidase activity of the conjugate. As a result of searching various biological components, it was found that collagen type I, type II, type III, type IV, type V, particularly type V has strong insulin binding activity.

[0008] The insulin binding active site of collagen is
Collagen molecules were physically and chemically and enzymatically fragmented and isolated, and the polypeptides containing insulin-binding activity were identified using the above peroxidase-labeled insulin. In other words, collagen type V degradation polypeptides, such as collagen type V alpha ₁ chain, or a collagen type V is cyanogen bromide decomposition insulin binding active site in the polypeptide of the resulting molecular weight 30kd was found that it is contained. This polypeptide also has heparin binding activity and can be efficiently purified on a heparin column.

Next, we determined the 30 kd polypeptide to determine the insulin binding site in the 30 kd polypeptide.
Enzymatic degradation of the polypeptide was performed. A 30 kd polypeptide endoprotease Glu-C [protease V
8: Boehringer Mannheim Yamanouchi Co., Ltd.] From the decomposition product, use a heparin column, ODS reverse phase column or the like to obtain 16 k
d polypeptide and 6 kd polypeptide can be collected.

[0010] Both of these polypeptides show heparin binding activity and insulin binding activity, respectively, but both activities are reduced as compared with the 30 kd polypeptide. 16kd
The polypeptide is further decomposed into three polypeptides by lysyl endopeptidase [manufactured by Wako Pure Chemical Industries, Ltd.], and these polypeptides can be fractionated by using an ODS reverse phase column. Although the entire amino acid sequence of collagen type V has not yet been determined, the amino acid sequences of the separated polypeptides and the 6 kd polypeptide were analyzed, and collagen X similar to collagen type V was analyzed.
Amino acid sequence of type I [Journal of Biological Chemistry (JBC), Vol. 263, No. 32,
17159-17166 (1988)], the structure of the 30 kd polypeptide and its amino acid sequence can be determined. As a result, the 6 kd polypeptide is bound to the C-terminal of the 16 kd polypeptide, and the amino acid sequence of the binding site is represented by the following formula (Formula 1):

[0011]

## STR1 ## -Gly-Gly-Arg-Gly-Thr-Hyp-Gly-Lys-Hyp-Gly-P
ro-Arg-Gly-Gln-Arg-Gly-Pro-Thr-Gly-Pro-Arg-Gly-Glu
-Arg-Gly-Pro-Arg-Gly-Ile-Thr-Gly-Lys-Hyp-Gly-Pro-

The amino acid sequence shown in the above was determined. 16k
By cleavage into the d polypeptide, a 6 kd polypeptide,
Since the insulin binding activity and the heparin binding activity are both decreased, the C-terminal portion of the 16 kd polypeptide and the N-terminal portion of the 6 kd polypeptide, that is, the amino acid sequence of the formula (Formula 1) are used to determine the collagen V type insulin binding activity and heparin This sequence is important for the expression of binding activity.

The collagen V-type degradation polypeptide referred to in the present invention may be any polypeptide having an insulin-binding active site containing the amino acid sequence of the above formula (Formula 1). In addition to chemical methods such as endopeptidase Glu-C degradation and enzymatic methods, it can also be produced by chemical synthesis methods and genetic engineering methods.

On the other hand, peroxidase-labeled insulin is also used for searching for an insulin-collagen binding inhibitor. That is, by reacting the sample and the insulin simultaneously with collagen type V and measuring the peroxidase activity of the collagen conjugate after the reaction, the insulin-collagen binding inhibitory activity of the sample can be assayed. The present inventors have found that insulin A-chain, B-chain, and insulin-B-chain degrading polypeptide have insulin-collagen binding inhibitory activity. The collagen-binding active site of insulin enzymatically fragments insulin, reacts the fragment and peroxidase-labeled insulin simultaneously with collagen type V, and inhibits binding of the insulin to collagen.
It is found by identifying collagen-binding insulin fragments. That is, they found that three polypeptides resulting from the degradation of insulin B chain by endoprotease Glu-C contain an insulin-collagen binding inhibitory active site.

The insulin B chain degrading polypeptide referred to in the present invention may be any polypeptide having a collagen binding active site contained in these three polypeptides.
In addition to the enzymatic decomposition method, it can be produced by a chemical synthesis method or a genetic engineering method. By the method of inhibiting insulin-collagen binding using these insulin A-chain, insulin B-chain, and insulin-B-chain degrading polypeptides, the operation of a composition for an insulin-binding carrier using collagen as a drag delivery system can be finely adjusted, Further, it can be used as a preventive or therapeutic drug for diseases caused by insulin function such as primary diabetes at the time of surgical invasion.

The insulin binding carrier provided by the present invention may be used as such or in the form of a conventional pharmaceutical dosage form such as a liquid, lotion, ointment, gel, tablet or capsule, or a solid substrate, It is used in the form of being attached to, bound to, or entangled with a transdermal device, a graft, or the like.

[0017]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Insulin-binding carrier bovine collagen type I, type II, type III, type IV, type V [Co., Ltd.]
Was dissolved in a 150 mM acetic acid / 50 mM NaCl solution to a concentration of 180 μg / ml, and diluted stepwise, and 50 μl thereof was dispensed into a 96-well microplate. After leaving at 37 ° C. for 2 hours, each well was washed with phosphate buffered saline (PBS (−)) containing no divalent metal ion, and then blocked using bovine serum albumin (BSA). Next, peroxidase-labeled insulin (manufactured by Sigma) was dissolved in PBS (-) to a concentration of 5 μg / ml, and 50 μl was dispensed into each well.
After reacting for a period of time, non-bound peroxidase-labeled insulin is washed and removed, and the peroxidase activity of the insulin / collagen conjugate is reduced to 2'-azino-di [3-ethylbenzthiazoline-sulfonate (6)] [ABT
S] and hydrogen peroxide as substrates.

As a result, it was observed that insulin was bound to each collagen. The results are shown in FIG. 1 is 180, 60, 20, 6.6, and 2.2 μm.
FIG. 4 is a diagram showing the binding of insulin at an absorbance at 414 nm (vertical axis) due to peroxidase activity at each collagen concentration of g / ml (μg / ml, horizontal axis).

Example 2 Insulin-Binding Polypeptide The above-mentioned collagen type V was dissolved at a concentration of 1 mg / ml, 10 μl of this solution was separated by SDS polyacrylamide electrophoresis, and transferred to a nitrocellulose membrane by Western blotting. After that, the mixture was reacted at room temperature for 2 hours in a 0.05% Tween 20-PBS solution containing 5 μg / ml peroxidase-labeled insulin. Peroxidase activity bound to the nitrocellulose membrane was detected using 4-chloro-1-naphthol and hydrogen peroxide as substrates. As a result, peroxidase-labeled insulin was observed to bind to alpha ₁ chain of collagen type V. Next, collagen type V was dissolved in a 70% formic acid solution containing 20 mg / ml bromocyanine to a concentration of 5 mg / ml, and reacted at room temperature for 24 hours to fragment collagen type V. The collagen type V fragment was subjected to electrophoresis and peroxidase-labeled insulin treatment in the same manner as described above. Insulin binding activity was observed in a fragment (30 kd polypeptide) having a molecular weight of about 30 kd.

The above-mentioned collagen V-type bromcian-treated product was subjected to high-performance liquid chromatography using a heparin column [TSK gel heparin 5PW: manufactured by Tosoh Corporation] equilibrated with 2M urea-PBS (-). After washing the column with an excess of the solvent, the 30 kd polypeptide was eluted with a solvent to which 1M salt was added, and then dialyzed under running water to remove the solvent, followed by lyophilization. 0.2M for this sample
It was dissolved in an ammonium acetate (pH 4.0) solution to a concentration of 0.1%, and 20 μg of endoprotease Glu-C was added to 1 ml of the solution, followed by reaction at 37 ° C. overnight. Next, the reaction product was applied to a heparin column equilibrated with 3-fold diluted PBS (-), and the column was washed with an excess solvent. The adsorbate was eluted with a solvent to which 0.5 M sodium chloride was added. Eluted O 2 equilibrated with 0.1% trifluoroacetic acid
When high-performance liquid chromatography using a DS-reverse phase column was performed and gradient elution was performed with 0% to 60% acetonitrile, fractionation was performed into two components. These two components are a fragment with a molecular weight of about 16 kd (16 kd polypeptide) and a fragment with a molecular weight of about 6 kd (6 kd polypeptide). When a dot blot using peroxidase-labeled insulin was performed, 3
Insulin binding activity was slightly weaker than that of the 0 kd polypeptide.

Lysyl endopeptidase digestion of the 16 kd polypeptide was performed to determine the amino acid sequence of the 30 kd polypeptide. Lysyl endopeptidase 1
It was dissolved in 50 mM ammonium bicarbonate (pH 7.8) to a concentration of 0 μg / ml, and the 16 kd polypeptide was degraded at 37 ° C. overnight. Perform high performance liquid chromatography using an ODS-reverse phase column in the same manner as described above,
Three fragments (L-1, L-2, L-3) were fractionated. Next, these were freeze-dried to remove the solvent. Next, these fractionated 6 kd polypeptides, L
The amino acid sequences of -1, L-2 and L-3 were analyzed using a protein sequencer 477A (Applied Biosystems). Amino acid analysis results and the aforementioned collagen X
Comparison of type I amino acid sequences, endoprotease Glu
As shown in FIG. 2 from the action point of -C, lysyl endopeptidase, the structure and amino acid partial sequence of the 30 kd polypeptide were determined. That is, FIG. 2 is a diagram showing the structure of a 30 kd polypeptide and its amino acid partial sequence.

Example 3 Insulin / Collagen Binding Inhibitor (Reference Example) In the same manner as described above, a 96-well microplate was coated with collagen type V and blocked with BSA. Next, 50 μl of a PBS (−) solution prepared so that peroxidase-labeled insulin was 5 μg / ml and insulin or insulin A chain or insulin B chain (manufactured by Sigma) was 100 μg / ml, was added to each well.
The reaction was performed at 37 ° C. for 2 hours. After completion of the reaction, unbound substances were washed and removed, and bound peroxidase activity was measured using ABTS and hydrogen peroxide as substrates. As a result, it was observed that the insulin A chain and the insulin B chain had the ability to inhibit insulin collagen. The results are described below. Fig. 3 also shows the results of the insulin B-chain degrading polypeptide.
Shown in That is, FIG. 3 is a diagram showing the ability of each insulin-related substance (horizontal axis) to inhibit insulin-collagen binding by absorbance at 414 nm (vertical axis) due to peroxidase activity. Next, 0.1% of insulin B chain, 50 mM
The above-mentioned endoprotease Glu-C (20 μg) was added to 1 ml of a phosphate buffer (pH 7.5) and reacted at 37 ° C. for 2 hours. The reaction product was fractionated by high performance liquid chromatography using an ODS-reverse phase column. Insulin B chain degradation products have the following formulas (Chemical Formula 2) to (Chemical Formula 4):

[0024]

Embedded image Phe-Val-Asn-Glu-His-Leu-Cys-Gly-Ser-His-Le
u-Val-Glu

[0025]

Embedded image Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu

[0026]

Embedded image Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr

And eluted from the reversed-phase column in the order of formulas (2) to (4). The insulin-collagen binding inhibitory activity of each polypeptide was measured in the same manner as described above, and the insulin-collagen binding inhibitory activity was found for each polypeptide. The results are shown in FIG.

[0028]

As is clear from the above description, the insulin-binding polypeptide of the present invention used for insulin binding has a novel activity of binding to insulin. In addition, they show useful actions and effects in fields where medical significance can be found. In particular, it is applied to a new insulin preparation and a drug delivery system. It can also be used to suppress the growth of cells including cancer cells using insulin as a growth factor.

[Brief description of the drawings]

FIG. 1 is a diagram showing the insulin binding properties of each collagen molecule.

FIG. 2 shows a schematic diagram of a 30 kd polypeptide and a partial amino acid sequence thereof.

FIG. 3 is a graph showing the ability of an insulin-related substance to inhibit insulin-collagen binding activity.

────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu-shi, Shiga Central Research Laboratory of Takara Shuzo Co., Ltd. (58) Fields investigated (Int. Cl. ⁶ , DB name) ) A61K 47/42 A61K 7/00 A61K 38/28

Claims (2)

(57) [Claims] The present invention relates to a collagen type I, II, III, IV, V type, and a collagen V-degrading polypeptide containing an insulin-binding site and having an insulin-binding activity. An insulin-binding carrier comprising at least one selected protein or polypeptide as an active ingredient. 2. The collagen V-degrading polypeptide having an insulin-binding activity is a polypeptide having an insulin-binding activity having a molecular weight of 30 kd or a polypeptide containing the polypeptide, which is produced by a bromocyan degradation treatment of collagen V-type. Item 4. The carrier for binding insulin according to Item 1.