JPS6245529A - Novel collagenase inhibitor - Google Patents

Abstract

NEW MATERIAL:A collagenase inhibitor extracted from the culture liquid of mouse adenoma cell colon 26 and having a molecular weight of 20,000+ or -10,000 (by gel-filtration). It has the following properties. Adsorptivity, non-adsorptive to Con-A-sepharose (in 0.5M NaCl, 5mM CaCl2, 30mM Tris-HCl buffer solution at 7.8pH); Stability; stable to the treatment with 1mM-APMA (at 35 deg.C for 15min) and 0.2% SDS (at room temperature for 70min). USE:Remedy for corneal ulcer, rheumatism, wound, etc., and agent for preventing metastasis of cancer. It specifically inhibits animal collagenase. PREPARATION:The titled substance can be produced e.g. by carrying out the primary culture of mouse adenoma colon 26 in 10% FBS-McCoy 5A medium, culturing the cultured cells in McCoy 5A medium, dialyzing and freeze-drying the culture liquid and purifying the dried product by DE-52 cellulose column chromatography and ultrogel ACA54 column chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a new collagenase inhibitor.

(Prior Art) Collagenase from mammals is known to be an enzyme that causes various diseases. For example, it is one of the important enzymes involved in the destruction process of cartilage and joints in rheumatoid arthritis [for example, Arthritis and Rheumatism].
tism), Vol. 20, p. 1231, 1977, p. 3]. It is also one of the enzymes that cause periodontal ligament disease, corneal ulcer formation, cancer metastasis (M wound invasion), epidermal excoriation varicella, etc. [For example, American Journal of
Pathology), Vol. 92, p. 509.

1978, and The New England Journal of Medicine.
ndJournal of Medicine),
Volume 291, page 652.

1974]. In order to prevent or treat these diseases, it is desirable to inhibit the activity of the enzyme collagenase. Compounds produced by organic synthesis are being studied for this purpose (for example, JP-A-57-212157).

On the other hand, although there are many reports on the detection of collagenase inhibitor activity in supplementary mammals, there are very few cases in which the substance can be identified.
G.B. Stricklin et al. (G, P, 5trick
lin et, al.), The Journal of
Biological Chemistry ('rheJourna)
of Biological Chemistry
), 258 volumes.

12252 pages, 1983], human polymorphonuclear leukocytes [H.W.

Macartney et al.), :x--o
Tupia 7 - Journal of Biochemistry (E
ur, J., Biochem.

, vol. 130, p. 79, 1983)], cow, dental pulp [Kurizumi et al., Journal of Biochemistry (J,B
iochem, ), vol. 96, p. 595, 1984
There have been reports of collagenase inhibitors since 2010. By the way, in order to develop this inhibitor for pharmaceutical use, it is desirable to produce it in large quantities using cancer cells, etc., which proliferate indefinitely. There have already been reports about collagenase inhibitors produced by cancer cells [D.B. De Vore et al.
et, al.), xubermental cell biology (Expl, Ce11.Biol,), 48
Vol. 367, 1980; N.N. Hatsuka et al.
(A,N,Halaka et,at,),Journal Opu Neurosurgery (J,Neurosu)
rg), Vol. 59, p. 461, 1983] The physical properties of the 1 inhibitor were purified to the extent that they were specified, and the properties fI
: I haven't researched it, and there's no mention of its usefulness.

(Problems to be Solved by the Invention) Among the conventionally known collagenase inhibitors derived from four mammals, the one that has been isolated from the tree and identified is the fibroblast of human skin, as described above. There have only been reports of cells, human polymorphonuclear leukocytes, bovine dental pulp, etc.;
In this regard, it is essential to be able to produce this substance in large quantities.

There are drawbacks to using the aforementioned fibroblasts, polymorphonuclear leukocytes, dental pulp, etc. as raw materials for collagenase inhibitors. How to overcome this point.

It is desirable to use cells that can increase N infinitely, but to date, there have been no reports of RL production and investigation of the properties of such cell-derived inhibitors to the extent of specifying their physical properties. Research and development was urgently needed.

(Measures to solve the IBj problem) Therefore, as a result of intensive research, the present inventors have developed a method for culturing mouse adenocarcinoma cell colon 26, which can be prepared in large quantities in order to proliferate indefinitely. We discovered a collagenase inhibitor that is expected to be effective in completely curing corneal ulcers, and completed the present invention.

That is, the present invention is a congenase inhibitor having the following properties.

(a) Molecular weight 20.000±io, ooo (gel filtration method) (b) Co
Does not adsorb to nA-Sepharose (0,5M NaCl,
5mM CaC! , , 50 mM Tris-H
Cl buffer, p) (under conditions 7,8).

[C) Good inhibition of animal collagenases from bovine dental pulp, bovine supinated sac, bovine cornea, chick skin, human leukocytes, etc.

(d) Does not inhibit bacterial collagenase.

(e) No cross-reactivity with bovine pulp collagenase inhibitor %A antiserum.

(f) 1 mM APMA treatment (35°C, 15 minutes)
It does not become inactive at all.

(g) Stable when treated with 0.2% SDS (room temperature, 70 minutes).

In the method for producing the collagenase inhibitor of the present invention, first, a medium used in normal cell culture is appropriately used.

Fetal bovine serum (Fe ta IBovi) was added at the early stage of culture.
ne Serum (hereinafter abbreviated as FBS) shows good growth when about 10% of the cells are added, but since there is a possibility that collagenase inhibitor is mixed in FBS, once the cells have grown to some extent.

It is preferable to culture in Yumo medium without FBS. The collagenase inhibitor of the present invention is prepared from the culture supernatant obtained by culturing in this manner by a method commonly used for protein purification. For example, adsorption methods using carriers, ion exchange methods, fractional precipitation methods, gel filtration methods, electrophoresis methods, various affinity chromatography methods, 1A7'5 methods using specific antibodies, etc. are desirable, and these methods can be used alone or in combination. .

Furthermore, to give an example of a specific culture and purification method, Matsukoi 5A containing mouse adenocarcinoma colon 26i and 10% FC8i
Medium (Shubei Munemura, Cell Culture Manual, p. 116, 1
982, Kodansha) and culture in monolayer until just before confluence. Next, the culture plate is continued to be cultured in a Fe2-containing catfish solution, and the culture solution is replaced and collected every two days. The culture solution was dialyzed against distilled water, lyophilized, and purified using 1DE-52 cellulose column chromatography and then Ultrogel ACA54 column chromatography.

The physicochemical properties of the collagenase inhibitor of the present invention thus obtained will be explained below.

The collagenase activity was measured using the well-known method of K. Terato et al.
,).

445, p. 753, 1976]K Therefore, 14
The radioactivity derived from 14c released using O-collagen as a substrate was measured. That is, 14 cm collagen was intraperitoneally administered to guinea pigs with 100 μCi of 14 CU-glycine per animal, and 6 hours later, the mice were turned over, extracted, and purified. 0.2% of this 14cm collagen
solution (50mM Tris-HC4p H7,8.

0.2M NaC6,5mMcac4, 0.5M
glucose) 200 ttL K, 30 m
M Tris-HCl pH7,8,5mMCaC1,
9 enzyme solution dissolved in 0,2M NaCl buffer 30
Add 0 μt and react at 35c for 12 to 16 hours. Next, 500 μt of Geoky'j7'jr was added, one reaction solution was mixed well, and the reaction was completely stopped. Centrifugation (3
,00Orpm, 10 minutes) to remove insoluble matter, and
00μt supernatant was taken and the radioactivity was measured.

Collagenase inhibitor activity was measured by the method of Kisumi et al. [
J, K15hi and T, Hayakawa;
Em,,) Volume 96, page 595, 1984] by Gyonatsu Tomo. That is, 1 mM APMAI niaminophenyl mercurin acetate (Aminophenyl acetate)
mercuric acetate)) at 350,3.
A sample of bovine pulp collagenase and collagenase inhibitor treated for 5 hours and subjected to activity comparison is heated at 35C for 10 minutes, 14 (1-collagen) is added, and the activity is measured according to the collagenase activity measurement method described above.

Next, we will discuss the expected healing effect of this substance on angular M ulcers. According to Manabe (Reizo Manabe, Japanese Ophthalmological Bulletin, vol. 24, p. 381, 1973), if a rabbit's cornea is corroded with caustic soda, serious corneal ulcers will develop in one to two weeks.
At the same time, collagenase in the cornea is activated. Activation of this collagenase is said to be the cause of corneal ulcers. Cysti/, which has a collagenase inhibitory effect,
It is said to be effective in treating corneal ulcers. From this,
It can be said that the substance of the present invention, which has the effect of specifically inhibiting animal collagenase, is very useful in the treatment of corneal ulcers.

(Example) Examples of the present invention are shown below.

+11 Method for obtaining collagenase inhibitor 10 cells of mouse adenocarcinoma colon 26 Matsukoi 5 containing FBS gold
Culture using A medium. The amount of culture solution is 1 per dish.
〇-Used. Ditsch is Corning Shakyu diameter 10
A tissue culture dishna 25020i of 0.0 mm was used. 1
0 Ditshu VC1 Ditshu Todoroki I X 106
Inoculate about 100 cells and % 37℃, carbon dioxide gas in S section 4
Prepared at the same time. Culture using a gas culture device. Approximately 3 days after starting the culture, the cells grow to near confluence in the bottle, and then the cells are replaced with Matsukoi 5-nin's medium (which does not contain FBS), which is a new friend, and cultured for another 2 days. Collect the top of the culture solution, exchange the soil with a new Matsukoi 5AJ@ field, culture for 2 days, and collect the culture soil.

This operation was repeated further, and the culture liquid supernatant collected was approximately 3
Get 00d. This culture #: is thoroughly dialyzed against cold distilled water. After dialysis, freeze-dry.

The residue was washed with 30 m
Dissolve in M Tris-HCl weak buffer solution, p) (7,8).The supernatant after centrifugation (10,000?, 30 minutes) was compared with P1 in the same buffer.
e1lulose column (Whatman, 2.6φα
X10crn). Wash the column thoroughly with the same buffer and collect the collagenase inhibitor active fraction and concentrate by lyophilization. Concentrated sample 10dlLKB
Shakyu Ushitrogel ACA54 column (2.5crn
φX? 4.5 m), 0.5 M NaCl,
30mM Tris HC containing 5mM CaC1,lz
Elute with L buffer (pH 7,8). Figure 1 shows the elution pattern of Ultrogel ACA54 chromatography. The solid line represents the absorbance at 280 nm, and the solid line with black circles represents the collagenase inhibitor activity in terms of the degree of inhibition of collagenase. In this way, a fraction (1244U) having collagenase inhibitor activity of the present invention was obtained.

(2) Molecular Weight The above fractions were purified by column chromatography on Bovine Trogel ACA54, and bovine serum albumin (BSA) was used as a standard molecular weight marker.

molecular weight 68,000), ovalbumin (OA, molecular weight 45,000), myoglobiga (Mb, molecular weight 17)
．． 800)t- was used to estimate the molecular weight of the substance of the present invention. From Figure 1, the molecular weight of the substance of the present invention is 20.00.
[It was estimated to be 3±10,000.

(To 31 Con A-Sepharose) Suction M Nomoto Invention vtJ
(200U) was added to Con A-Sepharose (con A
An attempt was made to adsorb it on a column (2,6αφX 5 cm) of -5epharose (manufactured by Pharmacia). 0.5M NaCl, 5mM CaC4, 30m
After washing with M TrisHCl buffer (pH 7,8), the substance of the present invention did not adsorb to Con A-Sepharose at all and showed fco (4) stability.
It was treated with an aqueous solution of MA (aminophenyl mercuric acid) at 35° C. for 15 minutes, but the activity was not lost at all.

In addition, the substance of the present invention (0.2 [J) was added to 0.2 5DS (
It was dissolved in an aqueous solution of sodium dodecyl sulfate) and treated for 70 minutes at room temperature, after which the collagenase activity in the dental pulp was measured. At this time, a report by Nice-Clark et al. (S, C1a
In order to eliminate the influence of residual SDS, non-ionic surfactant was added to remove the influence of residual SDS. As an agent, Torit:yX
-100 (trjton X-100), Tween-2
0 (tween-20), Prizzy 35 (Br1j-
55) etc., but in any case,
No decrease in inhibitor activity was observed due to SDS treatment.

(5) Measurement of inhibitor activity Collagenase inhibitor activity of the substance of the present invention.

Measured using the method described above. Approximately 200 mU of pulp collagenase is used to react with 14C-collagen.

The substance of the present invention was added to the reaction solution at an amount of 0 to 160 μt (5 μy/10 μt), and the extent to which the substance of the present invention inhibited bovine dental pulp collagenase activity was investigated.

The results are shown in FIG. 2, and it is clear that the substance of the present invention strongly inhibits bovine dental pulp collagenase activity.

(61if! Confirmation by aerophoresis 20 μg of type I collagen, 10 mU of bovine pulp collagenase and 60 mU of the present substance were mixed together,
21c for 4 days, slab U electrobed dynamic analysis of 6% 5O8-polyacrylamide was carried out, and K.
A report by Weber et al. (K.

Weber et, al., Journal of Biological Chemistry (J, B, C8), 244
roll.

4406, p. 19tS9] and stained with Comassive Brilliant Blue R type.

, the results are shown in Figure 313. Lane 1 is type I collagen;
Lane 2 Uiffi co-j-gen + collagenase, lanes 5 and 4 are type I collagen + collagenase + the substance of the present invention, and lane 5 is a mixture of co-j-genase + collagenase of the present invention. It is clear that collagen degradation is completely suppressed by the substance of the present invention.

(7) Substrate specificity Method of Kisumi et al., Journal of Biochemistry (J, Biochem
, ), Vol. 96, p. 395, 1984], whether or not the substance of the present invention inhibits the activity of various collagenases prepared according to the method was investigated as follows. 100μt
of 0.2 M NaCL, 5 mM CaO, I
0.05 M Tris containing M glucose f
-HC4 buffer, 20011t of various active collagenases, 80 μt (1.5 U) of the substance of the present invention, 200 μt
No. 14 (-collagen) was mixed and reacted at 35 C for 16 hours to measure collagenase activity. The results are shown in Table 1.

It was clear that the substance of the present invention well inhibits animal collagenases such as those of bovine dental pulp, bovine supination sac, bovine cornea, chick skin, and human leukocytes, but does not inhibit bacterial collagenase.

Table 1 Effect of Collagenase Inhibitor from Colon 26 on Collagenase of Each Strain Collagenase Activity (mU) Control Addition of Inventive Substance Inhibition (@Bacterial Collagenase 193j 203.5
0 burn granulation collagenase 282,0
10,8 96.2 Bovine dental cartilage collagenase
170,2 40.1 76.4 Chick skin collagenase 37,8 0
100 human leukocyte collagenase 54.3
0 i 00 bovine pulp collagenase 203.0 B, 1 9
6.0 Bovine corneal collagenase 270,0 1
3.5 95.0 (8) Cross-reaction of bovine pulp collagenase inhibitor with specific antiserum Method of Yonezumi et al.
), vol. 96, p. 395, 1984], a cross-reaction with the substance of the present invention was carried out by the method described in the above-mentioned document using a specific antiserum for bovine pulp collagenase inhibitor, which showed no cross-reactivity. Nakatsu nine.

(Effect of the invention) As described above, the collagenase inhibitor of the present invention has the following effects:
It is an extremely useful substance as a treatment for corneal ulcers. Further, it can be used as a drug for treating rheumatism, a drug for treating wounds such as burns, a drug for preventing cancer metastasis, a drug for treating periodontal ligament disease, and a drug for treating epidermal excoriation varicella.

[Brief explanation of the drawing]

Figure 1 is a graph showing the elution pattern of the substance of the present invention on a Bovine Trogel ACA54 column, Figure 2 is a graph showing the inhibitory effect of the substance of the present invention on collagenase activity, and Figure 3 is a graph showing the inhibitory effect of the substance of the present invention on collagenase activity. 6 is a chart showing the results of 5DS-polyacrylamide electrophoresis. Inhibitor 〒(, al') 7 2. 3 4 Otsu\.

Claims (1)

[Scope of Claims] Collagenase inhibitor having the following properties (a) Molecular weight 20,000±10,000 (gel filtration method) (b) Co
Does not adsorb to nA-Sepharose (0.5M NaCl,
5mM CaCl_2, 30mM Tris-HCl buffer, pH 7.8). (c) Good inhibition of animal collagenases from bovine dental pulp, bovine dental cyst, bovine cornea, chick skin, human leukocytes, etc. (d) Does not inhibit bacterial collagenase. (e) No cross-reactivity with specific antiserum of bovine dental pulp collagenase inhibitor. (f) No inactivation at all after treatment with 1mM MAPMA (35°C, 15 minutes). (g) Stable when treated with 0.2% SDS (room temperature, 70 minutes).