JPH0147480B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel proteinaceous element that is a component of an insulin secretion enhancing active factor. More specifically, it is a component that promotes insulin secretion in the cells and culture supernatant of microorganisms belonging to the genus Bordetella and has a pharmacological effect of maintaining and regulating blood sugar levels normally, that is, an insulin secretion-enhancing active substance produced by the genus Bordetella (hereinafter referred to as , abbreviated as N-IAP), which has reduced side effects. N-IAP, a diabetes drug, has the characteristic pharmacological action of promoting insulin secretion and maintaining and regulating blood sugar levels at normal levels.As it is a high-molecular protein, it also stimulates the antibodies that are expressed when N-IAP is administered. Due to its production, N-IAP has the disadvantage of inducing anaphylaxis when administered again. That is, it is desired to provide a safer drug that has no side effects other than insulin secretion enhancing activity, which is effective in the treatment and prevention of diabetes, and in particular suppresses anaphylaxis. As a result of intensive research, the present inventors succeeded in isolating and identifying a novel proteinaceous element by dissociating N-IAP using a highly concentrated urea aqueous solution. Each of these isolated and identified novel proteinaceous substances alone has no insulin secretion-enhancing effect, but when each of these proteinaceous substances is administered separately, a surprising effect is observed.
It has the same insulin secretion-promoting effect and the effect of maintaining and regulating blood sugar levels as when IAP is administered, and furthermore, due to the antibody production that occurs when N-IAP is administered, re-administration of N-IAP can cause anaphylaxis. It was found that the drawbacks that induce this were significantly reduced. In other words, when taken alone, the insulin secretion-enhancing effect, which is effective for the treatment and prevention of diabetes, is inactive, but by administering each separately, a novel drug that has insulin secretion-enhancing activity and has significantly reduced side effects can be obtained. This achieved the separation and identification of proteinaceous elemental substances. The configuration of the present invention will be explained below. Insulin secretion-enhancing active substance Insulin secretion-enhancing active substance (hereinafter abbreviated as "N-IAP"), which is the starting material in the present invention
microorganisms belonging to the Bordetella genus known as pathogenic bacteria (B. pertussis, B. parapertussis, B. bronchiseptica), particularly preferably B. pertussis I phase (B.
pertussis phase I) in a solid medium or liquid medium, and is a proteinaceous substance obtained by collecting and purifying the cells or the medium. Advantageous collection and purification of N-IAP from bacterial cells and cultures can be achieved by one or a combination of solubility, chromatography, molecular sieve, electrophoretic and biological methods. It can be achieved by many fractional purification methods commonly used in the field,
Therefore, the present invention is not limited to any particular collection and purification method. Column chromatography can be presented as an example of a highly advantageous collection and purification method for N-IAP. In this case, the culture supernatant of the microorganism is passed through a column made of packing materials of hydroxyapatite (manufactured by Seikagaku Corporation) and carboxymethylcepharose CL-6B (manufactured by Pharmacia Fine Chemicals). N-IAP purified by gel filtration using Cephacryl S-200 (manufactured by Pharmacia Fine Chemicals) is highly selectively adsorbed onto these columns, and then 0.5M
The purified product is eluted with an appropriately selected eluent such as 0.1 M phosphate buffer (PH 7.0) containing NaCl. By appropriately subjecting this purified product to dialysis, unnecessary salts are removed and highly active N-IAP is produced.
is obtained. In addition, since N-IAP is also present within the bacterial body, if desired, it can be added to the bacterial cell suspension, for example.
It is also possible to adopt a method in which N-IAP is leached from the bacterial cells during dissolution by adding NaCl. The Bordetella microorganism that produces N-IAP is
As mentioned above, Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are well known, but on the other hand, these pathogenic bacteria can be treated by conventional methods such as changing the culture medium composition, irradiating various types of radiation such as ultraviolet rays and X-rays, or applying mutagenic agents. Mutant strains obtained by mutating by various means also have N-IAP
It is useful as a producing bacterium. As a culture method, a liquid shaking culture method is preferred in terms of activity and yield, but other methods may also be used. The mycological properties and culture conditions of microorganisms belonging to the genus Bordetella are provided in Bergy's Manual of
Determinative Bacteriology 8th edition 1974 Baltimore: The Williams &
Willkna CO., J Exp Med. 129: 523-550.
(1969), Bacteriology Practice Summary: 3rd edition, page 6 et seq., 1972 (published by Maruzen Co., Ltd.), etc. Next, various physical properties of N-IAP will be explained in detail. State of existence and solubility characteristics: After desalination, the powder obtained by freeze-drying is a non-deliquescent white or light brown powder with a concentration of about 3-5 mg/ml.
Dissolves in water at room temperature, forms an insoluble white precipitate in 6NHCl, and dissolves in pyridine, sodium dodecyl sulfate,
Dissolve mercaptoethanol in cysteine solution. When cold (4°C), add ammonium sulfate to the solution of purified active substance,
Addition of dry ice, acetone, ethanol, trichloroacetic acid, zinc chloride solution, and solutions containing several other metal ions cause clouding and precipitation, respectively. water and chloroform or n
- In a butanol mixture, it becomes insoluble and collects at the interface between the two liquids. When an aqueous solution of N-IAP is heated above 80℃, it becomes cloudy. 0.1M phosphate buffer containing 0.5MNACl (PH7.0)
When this N-IAP is dissolved and then dialyzed using distilled water as an external solution, it becomes cloudy temporarily, but as the dialysis continues, it is completely redissolved and the cloudiness disappears. In addition, for high concentration solutions, 0.01M acetate buffer (PH4.5)
If thoroughly dialyzed, it may become pale brown in color and dissolve. Molecular weight: 10-30% density gradient acrylamide gel 37:
With a crosslinking ratio of 1, the pH of the concentration gel is 6.8 and the pH of the electrophoresis gel is 6.8.
After treatment with plate method SDS-polyacrylamide-disk electrophoresis method (Lumley method) in which pH is 8.8 and electrophoresis voltage is 90 V for 16 hours, 1
time and then staining with Coomassie blue. In order to convert the protein into SDS, treat with 1% SDS at 100°C for 5 minutes. N-IAP by SDS-gel electrophoresis
The molecular weight of is 105700±5000. Composition: Contains over 98% protein by weight according to the Lowry method. still,
The method for measuring each component was based on the following documents. Protein Lowry, OH, NJ Rosebrough, ALFarr,
and RJRandall. J.Biol.Chem.193:265 1951 Amino acid composition and composition ratio of protein components (μM/100μM; 6NHCl
Hydrolyzed at 110℃ for 24 hours, analyzed using Hitachi-835 high-speed amino acid analyzer): Aspartic acid (Asp) 7.5-7.9, Threonine (Thr) 6.8-7.8, Serine (Ser) 5.9-7.6, Glutamic acid (Glu) 8.8 ~9.4, Proline (Pro) 5.5~
6.4, Glycine (Gly) 8.7-9.6, Alanine (Ala)
9.1-10.8, cystine/2 (Cys/2) 1.0-2.0, valine (Val) 6.6-7.6, methionine (Met) 2.5-
3.3, isoleucine (Ile) 3.6-4.1, leucine (Leu) 7.5-8.7, tyrosine (Tyr) 5.1-6.6, phenylalanine (Phe) 3.7-4.5, lysine (Lys)
3.1-4.4, histidine (His) 0.9-1.4, and arginine (Arg) 6.1-6.6. Isoelectric point PH (migrated by polyacrylamide gel isoelectric focusing method, electrophoresis pattern and proportional measurement with various standard proteins): 9.3 ± 0.2 Disc electrophoresis pattern: Acrylamide gel (polyacrylamide concentration
7.5%, 1NKOH-glacial acetic acid buffer (PH4.3)), sample
In disk electrophoresis under the conditions of 30 μg, 4 mA current, 2 hours/1 gel, staining with Amido Black 10B, and decolorization with 7% acetic acid solution, the distance (based on the spacer/gel tip) was 2.3 ± 0.2 cm.
gives an extremely sharp single band at the position. Biological properties: It has insulin secretion-enhancing effects and glucose tolerance-improving effects in mammals, and these effects last for several weeks to several months after a single administration. Acute toxicity (LD ₅₀ ) is approximately 200μ in ddy mice (intravenous injection)
g/Kg body weight. Elemental Substance 1 It was confirmed that the N-IAP was fractionated and separated into two types of elemental substances by column chromatography or the like in the presence of a protein denaturing agent of 5M urea. As an example of an advantageous isolation and purification method for elemental substances,
An ion exchange method in the presence of 5M urea can be presented. That is, N-IAP is 3 to 5M, preferably 4M.
~5M urea presence PH5.4-6.8 preferably PH5.8-
The mixture is incubated in a 6.5 phosphate buffer for 12 hours to 96 hours, preferably 24 hours to 45 hours, and separated and purified using a CM-Sepharose column chromatograph equilibrated with the phosphate buffer. As a result, N-IAP was separated into two types of elemental substances with different molecular weights (hereinafter referred to as proteinaceous elemental substances CP-A and Ps-1, respectively, in descending order of molecular weight), and these elemental substances were separated using the same column. By regel filtration, it is obtained as a single disk electrophoretic substance. The elemental substance CP-A thus obtained does not exhibit insulin secretion enhancing activity by itself. It was also confirmed that CP-A is a basic protein. 2 CP-A alone hardly exhibits the desired insulin secretion enhancing activity. In other words, it is inactive in enhancing insulin secretion, but it is an elemental substance.
CP-A and another elemental substance Ps-1 are in
When mixed in vitro, N-IAP is reconstituted and further
Administer CP-A and Ps-1 separately, e.g. CP-
A was administered IV via the right hip joint at 3 μg/rat.
When Ps-1 was administered through the left hip vein after 8 hours, it exhibited insulin secretion enhancing activity equivalent to that of N-IAP, and the antibody production that occurred when administering N-IAP was significantly reduced.
- It was found that the incidence of anaphylaxis induced by re-administration of IAP was significantly reduced. 3 Physical properties of each elemental substance: (1) CP-A Molecular weight: SDS-gel electrophoresis 75000±5000 Basic protein amino acid analysis: Asp.6.4±0.7, Thr.7.3±0.8, Ser.8.2±0.9,
Glu.9.3±1.0, Pro.6.3±0.7, Gly.11.1±1.0,
Ala.9.0±0.9, Cys./21.2±0.3, Val.6.2±0.6,
Met.2.7±0.4, Ile.3.4±0.4, Leu.8.4±0.9,
Tyr.5.6±0.6, Phe.3.6±0.5, Lys.4.7±0.5,
His.1.1±0.3, Arg.4.9±0.5 Composition: 98% by weight or more of protein by Lowry method (2) Ps-1 Molecular weight: SDS-gel electrophoresis 28000±1200 Isoelectric point: PH5.8 Amino acid analysis Asp. 9.3±1.0, Thr.7.4±0.8, Ser.10.6±1.0,
Glu, 10.6±1.0, Pro.4.4±0.5, Gly.11.2±1.0,
Ala.10.6±1.0, Cys/2 1.3±0.3, Val.6.7±
0.7, Met.1.6±0.3, Ile.3.2±0.4, Leu.5.5±0.6,
Tyr.4.6±0.5, Phe.3.5±0.4, Lys.2.2±0.3
His.1.7±0.3 Arg.5.9±0.6 Composition: Example of protein content of 98% by weight or more by Lowry method Production and purification of N-IAP 1 Pertussis I phase bacteria Maeno strain IID 513 (Bordetella
pertussis phase ，Maeno Strain IID
513) (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) was cultured in a Brodet-Gengou plate medium at 37°C for 2 days, and then cultured in a modified Cohen-Wheeler medium supplemented with ion exchange resin, the composition of which is shown in Table 1 below. (CW medium) 200 ml was inoculated into a 500 ml shaken Kolben, and cultured with shaking at 37°C for 20 to 22 hours. The amount of bacteria in this culture solution was measured using a spectrophotometer (wavelength: 650 nm), and the ion exchange resin-added CW medium 1 was divided so that the amount of bacteria when added was a final concentration of approximately 0.1 × 10 ⁹ cells/ml. In addition to the shaken Kolben injected in step 2, shaking culture was performed at 37° C. for 48 hours (shaking frequency: 97 times/min). The mycological properties of the above-mentioned strain were consistent with the description in the literature regarding pertussis phase I mentioned above. Table 1 Cohen-Wheeler's modified medium composition Casamino acids 1.0g Yeast extract 1g Potassium dihydrogen phosphate 0.5g Soluble concentrated powder 2g 0.5% copper sulfate solution 1ml 1% calcium chloride solution 1ml 4% magnesium chloride solution 1ml Polypeptone 5g 1 % cystine solution 2.5 ml 0.5% iron sulfate solution 1 ml Sodium chloride 2.5 g (total volume 1000 ml with distilled water, 20% NaCH
After adjusting the pH to 7.2 with an aqueous solution, 3 g of an anion exchange resin (Diaion SA-20AP; manufactured by Mitsubishi Kasei Corporation) was added, and the mixture was sterilized with high-pressure steam at 121° C. for 15 minutes before use. ) The obtained 48-hour shaking culture solution was heated at 56°C for 30 minutes, and then centrifuged at 4°C (15,000 rpm) to separate the culture supernatant and bacterial cells. The obtained culture supernatant was used as a starting material for the purification and isolation of N-IAP. After adjusting the culture supernatant of 10 to pH6.0 with 1N hydrochloric acid,
As the first purification step, the mixture was passed through a hydroxyapatite column (2.5 x 4 mm) at a flow rate of 200 ml/hour. Most of the proteins were not adsorbed and passed through the column as they were, and the desired insulin secretion enhancing activity (see activity measurement method below) was hardly detected. The protein concentration is measured according to the notes in Table 2 below.
According to Lowry's method. Then, for the adsorbed substance, first 0.01M
Wash the column with phosphate buffer (PH 6.0), then increase the molarity of the phosphate buffer to 0.1 and PH to 7.0 to sequentially elute the adsorbed proteins. However, the target N-IAP was still not eluted under these conditions; it was further eluted with a phosphate buffer solution of the same composition containing 0.5 M NaCl in a phosphate buffer solution under the same conditions. Under these conditions, the target N-IAP could be efficiently recovered in agreement with the protein eluted. The obtained crude N-IAP was concentrated and then passed through a dialysis membrane with a permeability molecular weight limit of 8000 (Cat. NO. 3787 manufactured by Thomas).
-F25) and dialyzed twice against distilled water (total of 12 hours) and twice against 0.01M phosphate buffer (PH6.0) (total of 12 hours). In order to further purify, the solution containing N-IAP after the above dialysis was transferred to a carboxymethyl Sepharose CL-6B column (1.5 x 10 cm) equilibrated with 0.01M phosphate buffer (PH6.0).
passed through. There was no activity at all in the substances that were not adsorbed on this column, but then the molar concentration of the phosphate buffer was increased to 0.1, the pH was increased to 7.0, and the sodium chloride solution was further increased to 0.5M.
N-IAP was obtained by elution with an eluent containing . Since this portion still contains a small amount of impurity when analyzed by disk electrophoresis, the solution containing N-IAP is concentrated and subjected to gel filtration using Sephacryl S-200. The recovery rate, degree of purification, etc. of the activity of N-IAP purified through the above purification steps are shown in Table 2.

[Table] The purity of the substance obtained in the above final step was determined using polyacrylamide gel (polyacrylamide concentration 7.5%,
The assay was performed using 1N KOH-glacial acetic acid buffer (PH4.3) disk electrophoresis. The experimental method is the method of JVMaizel, Jr. (Biochem.Biophys.Res.Comm.1963, 13 483)
I went to bed. The sample per gel was 30 μg (as protein), the current was applied at 4 mA for 2 hours, the staining was performed with Amide Black 10B, and the decolorization was performed using a 7% acetic acid solution. 2 Pertussis I phase bacteria Higashihama strain IID 510 (Bordetella
pertussis phase I. Tohama Strain IID 510)
N-IAP was produced in the same manner as in Example-1 using a freeze-dried preserved bacterial strain (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University). Disk electrophoresis (using PH4.3 gel) of this product gave a single band, and the isoelectric point PH was 9.3±
It showed 0.2. The composition was about 98% by weight or more of protein, and the amino acid composition and composition ratio were as shown in Table 4.

【table】

[Table] Example Production and purification of elemental substances Purified insulin secretion enhancing active substance (N-
After dialyzing 10 mg of IAP) against 0.05M phosphate buffer (PH6.4) containing 5M urea for 24 hours, a CM-Sepharose CL-6B column (16φ×
200 mm) and elute with a sodium chloride density gradient using an aqueous solution containing 0.1 M phosphoric acid, 0.5 M sodium chloride, and 5 M urea at a flow rate of 2 ml/hour and 3 g/fraction. The results are shown in FIG. Through this operation, the insulin secretion enhancer was separated into two elemental substances with different molecular weights (named CP-A and Ps-1 in descending order of molecular weight as described above). In order to further improve the degree of purification of these components, the CP-A fraction was first concentrated and subjected to gel filtration using a Sephacryl S-200 column (16φ x 1100mm). This results in a pure
3.8 mg of CP-A was obtained. Elemental substances CP-A and Ps-1
Amino acid composition and composition ratio of protein components (μM/
100 μM) is as follows. CP-A Asp.6.4, Thr.7.3, Ser.8.2, Glu.9.3, Pro.6.3.
Gly.11.1，Ala.9.0，Cys.／２ 1.2，Val.6.2，
Met.2.7, Ile.3.4, Leu.8.4, Tyr.5.6, Phe.3.6,
Lys.4.7 His.1.1 Arg.4.9 Ps―1 Asp.9.3, Thr.7.4, Ser.10.6, Glu.10.6,
Pro.4.4，Gly.11.2 Ala.10.6 Cys.／２ 1.3，
Val.6.7, Met.1.6, Ile.3.2, Leu.5.5, Tyr.4.6,
Phe.3.5, Lys.2.2, His.1.7 Arg.5.9 3 Elemental substance CP-A isolated and purified in each of the above examples
As shown in FIG. 2, it was confirmed that Ps-1 and Ps-1 were a single substance by disk electrophoresis. Furthermore, the results of a comparative study of the insulin secretion enhancing active factor and the elemental substances CP-A and Ps-1 by SDS/disk electrophoresis are shown in FIG. In addition, the physical properties of the elemental substances CP-A and Ps-1 are as follows:
It is as detailed in the section of "Physical properties of elemental materials" above. In addition, the experimental conditions for each electrophoresis are similar to those in Examples. Examples Pharmacological effect 1 Insulin secretion enhancing activity measurement method The activity of each active substance can be measured by the reactivity of animals to various insulin secretion stimulating substances, but glucose is usually used as the stimulating substance. Γ Test animal Male Wistar rat (weight 130-140 g) Γ Test method The active substance of each potency is dissolved in physiological saline.
Inject 0.2ml into the femoral vein under ether anesthesia,
Days later, insulin secretion enhancing activity is measured. The subjects were fasted for 18 to 20 hours on the first day of the experiment. The measurement method is to collect 0.1 ml of blood from the tail vein of a rat, immediately administer 1 ml of 30% glucose solution per 100 g of body weight intraperitoneally, and after exactly 15 minutes, collect 0.1 ml of blood again. Insulin secretion enhancing activity is determined from the difference in blood glucose level and blood insulin level before and after glucose administration. Blood sugar levels are measured using the glucose oxidase method, and insulin is measured using the dual antibody method. The methods for measuring blood sugar level and insulin are based on the following literature and Kitt, respectively. Blood sugar level: Glucose oxidase method Bergmeyer, H.-U., and Bernet, E.in
“Methods of enzymatic analysis”
Bergmeyer, H.—U., eds, New York.
Academic press P 123 (1963) Glucostat Insulin: Two-antibody method Morgan, CR, and Razarow, A.Diabetes
12 115 (1963) Insulin Liaquit - Dynabot Co., Ltd. First, the ΔI/ΔG values of the rats in the active substance administration group and the control group are determined according to the following formula.

[Table] The reason why blood sugar levels are used to calculate activity is that the amount of insulin secreted is greatly affected by blood sugar levels. Next, the potency of the active substance is calculated as follows: Unit = (average ΔI/ΔG of rats in the substance-administered group)
- average ΔI/ΔG of control group/average ΔI/ΔG of control group) x 10
Find it using 0. The specific activity of each substance is determined by dividing the titer unit by the weight. 3. Insulin secretion-enhancing activity The insulin secretion-enhancing active substance isolated and purified by the present inventor promotes insulin secretion in mammals, exhibits the effect of maintaining and regulating blood sugar levels at normal values, and further enhances antibody production. It also shows the effect of enhancing cellular immunity. Since this active factor exhibits these effects in extremely small amounts, it is useful as a therapeutic and preventive drug for various types of diabetes, or as a drug for diseases caused by abnormalities in immune function (e.g., malignant tumors, aplastic anemia, rheumatoid arthritis, etc.) It is. The present inventor has discovered that N-
When IAP is separated into its component substances, its pharmacological action becomes inactive, but by separately administering each of these component substances, we succeeded in obtaining a new single substance that exhibits its pharmacological activity. did. elemental substance
Table 6 shows the insulin secretion enhancing activities of CP-A and Ps-1. 4. Side Effects and Toxicity The insulin secretion-enhancing active substance isolated and purified by the present inventor exhibits insulin secretion-enhancing activity in extremely small amounts, and is considered to be extremely useful as a therapeutic and preventive drug for various types of diabetes. However, apart from its original activity, this insulin secretion-enhancing active substance exhibits so-called side effects such as increasing the number of white blood cells and increasing sensitivity to histamine, and also exhibits strong antibody production, that is, antigenicity. It turned out. In particular, antigenicity may lead to serious consequences such as anaphylactic shots upon second and subsequent administrations. Since the insulin secretion-enhancing active substance shows its original effect in extremely small amounts, the possibility of this side effect occurring is thought to be low. It goes without saying that it is desirable to eliminate side effects without causing any side effects. The present inventor has determined that by separating an insulin secretion-enhancing active substance into component substances and administering each of these component substances separately, the insulin secretion-enhancing activity has the same level of activity as the insulin secretion-enhancing substance, and further It was confirmed that the antigenicity was reduced to less than 1/30 and the acute toxicity value (LD ₅₀ value) was also reduced.
Table 6 shows the antigenicity of the element substances CP-A and Ps-1. Γ Antigenicity measurement method The sample was administered to a group of 6 male Hartley guinea pigs (body weight 250 g) three times on consecutive days. On the 22nd day after administration, 1 mg of the same sample was intravenously injected, followed by an anaphylactic shot. The number of deaths was observed.

[Table] Table 7 shows the intravenous LP ₅₀ values for elemental substances CP-A and Ps-1.

[Table] Elemental substance CP-A is another elemental substance Ps-1
It is considered to be useful mainly as a diabetes treatment drug in combination with. Currently, the only drug treatments for diabetes include insulin injections and oral administration of hypoglycemic drugs, which are only symptomatic treatments, and it is safe to say that the disease is almost incurable. Moreover, there is the complication of having to go to the hospital every day for insulin injections.
When administering hypoglycemic drugs, the occurrence of an abnormal drop in blood sugar levels is always considered a danger. The characteristics of elemental substance CP-A are that when combined with another elemental substance Ps-1,
It not only has insulin secretion activity, but also has the ability to increase blood insulin and quickly return blood sugar levels to normal only when blood sugar levels are elevated under various conditions (hyperglycemic conditions, especially during glucose loads similar to feeding). Express. The advantage of administering the elemental substance CP-A separately from the other elemental substance Ps-1 is that the activity can last for several weeks to several months after a single administration;
-10397 and Japanese Patent Application No. 52-49641, the drawback of not being able to be administered continuously due to the development of antibody production has been overcome, and continuous administration has been made possible. .
Furthermore, the elemental substance CP-A is another elemental substance Ps
-1 can be used not only as a treatment for diabetes, complications of diabetes, and various adult diseases caused by diabetes, but also for pre-diabetic conditions, a tragic state for which there is currently no treatment. It is useful as a preventive and therapeutic agent and a diagnostic agent for juvenile diabetes. As detailed above, by combining the elemental substance CP-A with another elemental substance Ps-1,
It is extremely useful as a diabetes treatment and prevention drug and an immunomodulator, and the effective amount for the human body is approximately several 10 Units/Kg in solid form depending on the specific activity of the active substance.
(weight) to tens of thousands of Units/Kg (weight),
Approximately several 10 Units/Kg for immune-enhancing activity applications
(body weight) to tens of thousands of Units/Kg (body weight). For administration to patients, intravenous administration is the most effective for each active use, and other methods include intraperitoneal, intramuscular, and subcutaneous administration, as well as direct administration into the gastrointestinal tract, oral administration, intrarectal administration, sublingual administration, and intradermal administration. Administration to the nasal mucosa, artery, lymph or trachea is also effective. Examples of dosage forms for each active use include injections, suppositories, enteric/gastric solutions, sublingual tablets, and inhalants. To give an example of the simplest composition of an injection solution,
Insulin secretion enhancing activity 10000 Units, NaCl 9
mg and 1 ml with sterile distilled water. It will also be obvious to those skilled in the art that when formulating the drug, any other ingredients that do not impair the activity may be mixed.

[Brief explanation of drawings]

1 to 3 are explanatory diagrams of experiments of the present invention,
Figure 1 shows CM-cefmarose CL-6B column chromatography. Figure 2 is a Disc-electropherogram. Figure 3 is the SDS of elemental substances.
This is a dise electropherogram.

Claims (1)

[Claims] 1. Molecular weight by SDS-gel electrophoresis method is 75,000
±5000; The protein content is 98% by weight or more by the Lowry method, and the amino acid composition and composition ratio (μM/100μM) of the protein component are ASp.6.4±0.7, Thr.7.3±0.8,
Ser.8.2±0.9, Glu.9.3±1.0, Pro.6.3±0.7,
Gly.11.1±1.0, Ala.9.0±0.9, Cys./2 1.2±
0.3, Val.6.2±0.6, Met.2.7±0.4, Ile, 3.4±0.4,
Leu.8.4±0.9, Tyr, 5.6±0.6, Phe.3.6±0.5,
Lys.4.7±0.5, His.1.1±0.3 and Arg.4.9±0.5: and a basic protein, which is associated with the discovery of insulin secretion enhancing activity when combined with Ps1. CP-A. 2. Incubate the insulin secretion-enhancing active substance obtained by purifying a microorganism belonging to the genus Bordetella in a phosphate buffer (PH5.4-6.8) containing 3M to 5M urea for 12 to 96 hours, and then incubate it in a phosphate buffer (PH5.4-6.8) for 12 to 96 hours. PH
5.4-6.8) A method for producing a proteinaceous element CP-A, characterized in that separation and purification are performed using a CM-Sepharose CL-6B column equilibrated according to 5.4-6.8).