• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A novel biologically active substance named Islets-Activating Protein obtained from the cultivation of the miocroorganism Bordetella and having insulin secretion promoting action as well as glucose tolerance improving action for mammals.
    公开号:SU871721A3
    申请号:SU782576599
    申请日:1978-01-31
    公开日:1981-10-07
    发明作者:Ядзима Мотоюки；Хосода Конти；Томиока Чиканори；Уи Мичио
    申请人:Какеньяку Како Кабусики Кайся (Фирма)；
    IPC主号:
    专利说明:

    solid ammonium sulfate to reach a point that approaches the saturation point of the dissolved salt, and the pH is adjusted to 6-7 by the addition of dilute aqueous ammonia solution. Then the precipitate is washed with water, and the biologically active substance is extracted from it with a 0.1 m solution of Tris buffer with a pH value of 8.0 containing 0.5 M nttri chloride.
    The resulting biologically active substance is a non-spreading white or light brown product that dissolves in water at room temperature in a concentration of from 3 to 5 mg / ml. When ingested with bn, a solution of hydrochloric acid forms an insoluble white precipitate. It is soluble in pyridine, sodium dodecyl sulfate, 2-mercaptoethiol and cystine solution. Adding a mixture of dry ice with acetone or ethanol, trifluoroacetic acid or zinc chloride solution, or a solution containing some metal's metallic ions to a solution of purified biologically active substance per heath (at 4 ° C leads to the formation of a cloudy-white precipitate. When ingested a solution of water and chloroform or n-butanol, the biologically active substance does not dissolve and is collected in such a solution around the interface between two liquids.
    When an aqueous solution of a substance is heated to or above, it becomes cloudy-white. The ori dissolving the substance in 0.1 M phosphate buffer at pH 7.0) containing 0.5 M sodium chloride when dialysis using distilled water as the external liquid, the substance temporarily becomes turbid, and if dialysis is continued, the substance completely dissolved. If the highly concentrated solution is subjected to thorough dialysis using 0.01 M acetate buffer {pH-4.5.), Then this substance becomes light brown in color and dissolves. The molecular weight of the substance 77000 ± 6400 is determined by filtering the gel through a column of Biogel P-100, equilibrated with 0.1 M phosphate buffer, which contains 0.5 M sodium chloride.
    The protein content is greater than 95% by weight, and the saccharin content is 1% by weight. The lipid concentration is below the minimum detectable limit.
    The biologically active substance enhances the secretion of insulin and has an improved effect on maintaining glucose levels, and these actions persist for several weeks to several
    ec after received same
    ozy. Acute toxicity ld
    50 200 mg / kg live weight.
    Example 1. Getting biologically active substances. Bordeteu and pertussis lyophilized and preserved microorganism are cultured in plates using a Bordet-Jangui medium with a temperature of 37 ° C for 2 times, and then using platinum elite to inoculate these bacteria in a 500 ml flask, shake 200 ml of ion-exchange resin, odified medium Cohen-Weeler (medium KB), the composition of which is given S table. 1, added with a pipette, after which the culture was carried out by shaking for 20 to 24 hours at 37 ° C. The concentration of bacteria in the nutrient solution is measured using a spectrophotometer (with a wavelength of 650 nm); this solution is added to a two-liter shake culture flask, to which a 1 liter of ion exchange resin is added with a pipette, together with the KB medium introduced into it, due to which the final concentration of bacteria is approximately 0xlOx ad / ml and then the wire The cultivation process was shaken for 48 hours (the shaking frequency was 97 cycles / min at a temperature).
    When using this liquid medium, it is added together with distilled water, taken in an amount sufficient to bring the total volume up to 1000 ml, and after bringing the pH to 7.2 by adding a 20% sodium hydroxide solution to the solution of the medium, additionally adding 3 g of anion exchange resin are applied, and then the whole mixture is autoclaved at 121 ° C for 15 minutes.
    Prepared after 48 hours of shaking, the culture solution was subjected to aging at a temperature of 56 ° C for 30 minutes and then centrifuged (at a rotation speed of 15000 rpm) at a temperature of 4 ° C to separate the upper layer and the bacterial cell mass, after of which the thus-obtained upper layer of the medium solution is used in. as a starting material for the purification and isolation of the target active substance.
    10 liters of this top layer after adjusting the pH to 6.0 by adding 1 n. The hydrochloric acid solution is introduced into a hydroxylapatite column (2.5 x 4 cm) at a flow rate of 200 ml / h in the first purification stage.
    Most of the protein passes through the column without adsorption in it, and
    target secretory insulin activity is difficult to determine. Protein concentration is measured by the method of Lowry.
    To determine the adsorbed substances, the column is first washed with 0.01 M phosphate buffer (at pH 6.0) and then (after increasing the molar concentration of phosphate buffer to 0.1 and pH to 7.0), the adsorbed (elu. The conditions do not eluir cosiness the target biologically active substance. Additional elution is carried out using phosphate buffer of the same composition, but containing 0.5 M sodium chloride. Under these conditions, the target substance can be isolated with high efficiency in accordance with the eluted protein.
    The obtained biologically active substance I is concentrated and, after being placed in a dialysis membrane with a maximum molecular weight for penetrating molecules of 8000, dialysis is carried out twice (for a total of 12 hours) with distilled water and twice (for a total of 12 hours). using 0.01 M phosphate buffer (with pH 6.0) -. For further purification, the solution containing the dialyzed biologically active substance is passed through a column (1.5 x 10 cm) with carboxymethyl sepharose CL-6B, equilibrated with 0.01 M phosphate buffer (pH 6.0). Products that were not adsorbed in this column show no activity. Then, after increasing the molar concentration and the pH of the phosphate buffer, respectively, to 0.1 and 7.0, similar elution was performed by adding 0.5 M salt, resulting in a target biologically active substance which was consistent with the eluted protein.
    Since this product still contains a small amount of impurities in the disk electrophoretic direction, this substance is further concentrated, and after being placed into the dialysis membrane, it is dialyzed twice (for a total of 12 hours) with distilled water and twice (for a total of - within 12 hours) using 0.01 M phosphate buffer (pH 7.0), and then the dialyzed sample is passed through a column (1.5x8 cm) with KonA syopharose 4B, which is equilibrated with 0.01 M phosphate buffer ( with pH 7.0).
    As a result of the appearance of the same buffer, minor traces of protein are eluted; however, this protein component does not have any activity. Further elution.
    A 0.1 M phosphate buffer (pH 7.0) containing 0.5 M sodium chloride g provides a substance that corresponds to the eluted protein.
    Since this part still contains an insignificant amount of impurities in the disk electrophoretic direction, this protein component is collected, condensed and decomposed using
    0 0.1 M phosphate buffer (pH 7.0), which contains 0.5 M sodium chloride, after which the dialyzed sample is subjected to gel filtration by passing through a column
    5 (2.8x60 cm) with F-100 Biogel, balanced by the same sam buffer. As a result of this, a pure biologically active substance is obtained, which corresponds to the protein
    0 component, characterized by the presence of a peak at the level of molecular weight of about 80,000.
    In tab. 2 presents data characterizing the degree of purification of the drug.
    five
    The degree of purity of the substance is determined in accordance with the method of disk electrophoresis using a polyacrylamide gel (polyacrylamide concentration - 7.5%, buffer 1N potassium hydrate - ice on acetic acid with pH 4.3).
    The amount of sample per gel is 30 µg (in terms of pa protein),
    5 wherein the experiment was carried out with the application of a current of 4 mA for 2 hours, with the formation of a spot of amide black 10 V and the removal of stains using a 7% acetic acid solution. The sample obtained in this last stage is the only substance that is completely free of impurities, and the activity in terms of accelerating insulin secretion, which is determined by
    5 in the course of the experiment, corresponds to the activity of the isolated substance. To establish the detailed structure of the biologically active substance, it is subjected to the following
    0 VAT (sodium dodecyl sulfate) polyacrylamide gel electrophoresis.
    A sample of biologically active substance in the amount of 50 µg / tube (in terms of protein) is added to the mixture of 1% VAT, 1% 2-mercaptoethanol and 4 mol of urea, and after two hours of incubation at 37 ° C, the mixture is placed in 1% - polyacrylamide gel, which contains 1% VAT. Then, after four hours of current passing 8mA / gel, a spot is obtained using Coomassol, followed by removal of the spot.
    5 7.5% acetic acid. Example 2. BordeteKfa pertussis lyophilized and preserved microorganism (strain 1H 114 / 3779B /) was cultivated by shaking followed by isolation of the analog of example 1, resulting in 10 l of the surface layer of the culture in a liquid medium. The surface layer thus obtained is further divided into two parts, one of which is passed through a hydroxyapatite column (2.5x4 cm) with a flow rate of 200 ml / after adjusting the pH of the surface layer to 6.0 by adding 1N. hydrochloric acid. This column is washed with water, and then the adsorbed substance is eluted with 0.1 phosphate buffer, which contains 0.5 M sodium chloride (pH 7.0). The eluate thus obtained is placed in a dialysis membrane with a permeability for substances with a maximum molecular weight of 8000 and dialysis is carried out three times using distilled water (for a total of 18 hours), resulting in a biologically active substance. After that, the obtained substance is subjected to freeze-drying treatment, as a result of obtaining a fine brownish-white powder that accelerates the secretion of insulin. . Ammonium sulfate is added to a 90% saturation level in the other 5 l of the surface layer of the culture in a liquid medium (pH is adjusted to 6.5 by adding a weak aqueous solution of ammonia), resulting in all protein substances precipitating. Then the precipitate is completely washed with water and leached with 0.1 m Tris-0.5 sodium chloride buffer (pH 9) to obtain an extractive solution. This prepared extract solution is first neutralized with 1N. solution of hydrochloric acid, and then subjected to a three-fold dispute (for a total of 18 hours) using distilled water and the above-mentioned dialysis membrane to obtain a biologically active substance, resulting in 11.2 mg of freeze-dried poro (sample B) . Example 3. With the use of a lyophilized and preserved microorganism BordeteWa pertussis (strain Maepo, phase 1), the experiment described in example 1 is repeated, resulting in 23.6 mg and 10.8 mg of each of the lyophilized powdered samples of biologically active substances ( samples C and D). The specific activity of each sample is given in Table. 3. Acute toxicity (IDjQ) for each sample when administered intravenously to mice is given in Table. 4. Thus, powdered samples of biologically active substances can be used as medicinal or prophylactic agents for diabetes, and their effective dosage is in the range of 1-100 µg / kg of live weight. Example 4. The microorganism Bordeteje and pertussis (strain To ma, phase 1) is subjected to cultivation. analogy of example 1, and the upper layer of the culture in a liquid medium, prepared by isolation from the microorganism, is used as a starting material. Acetone, which is pre-cooled with dry ice, is added dropwise to 100 liters of shaken surface layer, cooled with dry ice to a final concentration of 60%. After that, the precipitate is collected with continuous separator centrifuges (speed is 500 rpm, temperature 4s). The residue thus obtained was dried to obtain a product, which was dissolved in 500 ml of 0.01 M phosphate buffer (pH 6, b), and then passed through a column (5x4 cm) with hydroxylapatite. The biologically active substance is accumulated in a component that is eluted with a 0.1 M formate buffer (pH 7.0) containing 0.5 M sodium chloride. The eluted substance is collected, condensed and dialyzed using 0.01 M phosphate buffer (pH 6.0), and then passed through a CL-6B CM-Sepharose column (2f5x25 cm), which is balanced biologically with the same buffer. The active substance is eluted with 0.1 M phosphate buffer containing 0.5 M sodium chloride. Subsequently, the biologically active substance eluted in this way is collected, condensed and dialyzed using 0.1 M phosphate buffer (pH 7.0) containing 0.5 M sodium chloride. After that, the substance is placed in a column with Biogel P-150 (2x105 cm), which is balanced with 0.1 M phosphate buffer containing 0.5 M sodium chloride and 4 M urea. This biologically active substance is a protein that is characterized by nal -. The peak has a molecular weight of about 80,000 and 38 mg of its lyophilized powder is obtained. The product thus obtained is characterized by the following chemical composition: 95 wt.% And more protein, 1-2 wt.% Carbohydrates, and no lipid was detected.
    The specific activity of the product is 930 U / µg.
    Example 5. Bordete t a pertussis lyophilized and preserved microorganism (strain Toma, phase 1) is cultivated by shaking by analogy with Example 4, and isolating the bacterial cell mass from a culture in a liquid medium results in 100 l of the surface layer. A 50% aqueous solution of zinc chloride is added to the resulting surface layer with shaking at room temperature until the pH is adjusted to 6.0 (final concentration 1%). This precipitate is dissolved in 10% sodium phosphate, then placed in a dietary membrane, dialyzed with water, followed by equilibration with 0.01 M phosphate buffer. Then this solution is passed through a column (10x5 with hydroxyapatite, then washed with 0.1 M phosphate buffer (pH 7.0), followed by elution with 0.1 M phosphate buffer containing 0.5 M sodium chloride. The eluate is condensed and pass through a column (2.5x100 cm) with Sephacryl S-200, and then elute with 0.1 M with phosphate buffer (pH 7.0), which contains 0.5 M sodium chloride and 4 M urea. gel filtration to obtain a biologically active substance.
    The molecular weight of the obtained material is approximately 7,200 (by gel filtration), and 40 mg of a dry powder is obtained. The resulting product is characterized by the following chemical composition of approximately 95% by weight or more protein, 1-2% by weight of carbohydrates, and no lipid was detected. The specific activity was 890 U / µg.
    Example 6. BordeteCfca pertussis microorganism (Toma strain, phase 1 is cultivated in Bordet – Zhangu medium at a temperature for 2 days, and then in a beveled Bordet – Zhangou medium at a temperature of 37 ° C for 20–24 ° C, followed by using a platinum loop, these bacteria inoculate a modified Coen-Weeler solid medium with added charcoal (KB), the composition of which is shown in Table 5, followed by cultivation for 48 hours at a temperature of 5 kg of the obtained moist bacterial cell mass. in 5 l istillirovannoy water and maintained therein at a temperature of 56 ° C for 1h.
    After cooling, thimerosaE- is added to the suspension until its final concentration reaches 0.01% and its
    centrifuged (rotation speed 15000 rpm) for 30 minutes to separate the bacterial cell mass. The collected cell mass is suspended in distilled water, which contains 4 mo of urea and 1 mole of sodium chloride, and then subjected to ultrasonic treatment at a low temperature () to destroy live bacterial cells.
    The resulting suspension was centrifuged (at a rotational speed of 15,000 rpm) for 30 minutes, and the sediment-free surface layer was dialyzed using water, followed by equilibration with 0.1 M phosphate buffer (pH 0.6), and then passed through a column (10x5 cm) with hydroxyapatite. After saturation of the adsorbed rimes with 0.01 M phosphate buffer (pH 0.6) and 0.1 M phosphate buffer (pH 7.0), the target substance is eluted with 0.1 M phosphate buffer (pH 7.0), which contains 0.5 M sodium chloride.
    The biologically active substance thus obtained is equilibrated with 0.01 M phosphate buffer (pH 6.0) and then passed through a column (2.5x30 cm) with CM-Sepharose CL-6B. This column is first passed through with 0.05 M phosphate buffer pH 6.0 and then eluted with 0.1 M phosphate buffer (pH 7.0), which contains 0.5 M sodium chloride, with elution of the biologically active substance. This eluted substance is then condensed: fl and dissolved in 0.1 M phosphate buffer (pH 7.1), which contains 4 M urea and 0.5 M sodium chloride, and then passed through a column (2.5 x 115 cm with Sephacryl). S-200.
    After this operation, solvent was eluted to obtain 40 mg of the desired substance.
    Based on the results of and :::.; Eracium by gel filtration, it is calculated that the molecular weight is 74,000, and the product is characterized by the following chemical composition: over 95 wt.% Protein, 2 wt.% Carbohydrates, and no lipid was detected. The specific activity is 920 U / µg.
    The composition of the medium KB is given in table.5. (When using this solid medium, it is dissolved by heating after adjusting the pH to 7.2, and then 5 g of powdered wood coal is added to the medium and subjected to autoclaving at a temperature for 20 minutes),
    As an efficient preparation of a biologically active substance, there can be various chromatographic treatments. Implementation of such
    The method, with its appropriate inclusion in the sequential series of operations of the preparation process, as described in Example 1, makes it possible to partially eliminate the preparation stages. Subsequently, both the purification and the preparation, when carrying out such a method, is carried out in analogy to that described in Example 1. This method is described in more detail below.
    Example 7. The lyophilized and preserved microorganism BordeteBSa p3rtussis (strain Toma, phase 1) is cultivated (as described in example 1). 10 l of the obtained surface layer of the culture in liquid medium is passed through a column (column size 5x2 cm, material flow rate 60 ml / h) with ohydroxide and apolitanite, and then washed with 300 ml 0.01 M phosphate buffer (pH 7.0). After that, 0.1 M phosphate buffer (with pH 7.0), which contains 0.5 M sodium chloride, is passed through it with a flow rate of 15 ml / h to elute the biologically active substance. The resulting material is condensed to a volume of about 15 ml using Ficol-400 product followed by its fourfold dialysis for a total of 24 hours, using 2 liters of distilled water, and then it is subjected to fourfold dialysis again for a total of 24 hours of time using 1 L 0.01 M acetate buffer (pH 4.5), which contains 0.1 M sodium chloride or 0.1 M lithium chloride-hydrogen chloride (pH 4.5) for balancing. This product is passed through a p-acetoxymerutaniline-Sepharose BMV column (column size 1.2x8 cm), which is equilibrated with the same buffer as mentioned above, with a flow rate of 5 ml / h and after thorough washing with the same buffer, the adsorbed substance eluted with the same buffer, to which 0.01 ML -cysteine was added. The resulting substance was collected and then condensed to a volume of approximately 5 ml using the product Ficol-100 with a subsequent threefold dialysis for a total of 12 hours using 0.1 M pho fatnogo buffer (pH 7.0) in an amount of 2 liters, which contains 4 mole of urea and 0.5 moles of sodium chloride to equilibrate, after which it was subjected to further equilibration using the same buffer. Gel filtration was carried out in a Sephacryl S-200 column (2.8x95 cm).
    The biologically active substance is obtained as a product with one peak of molecular weight 65000 ± 7000
    (as further defined by the expiration of the reference protein), coinciding with the peak of activity. This substance is dialyzed (using 2 liters of distilled water, 5 times for a total of 48 hours), and then lyophilized to obtain 2.1 mg of white powder.
    This product is characterized by a single band in polyacrylamide gel electrophoresis (with a gel pH of 4.8) and the following chemical composition: over 98 wt.% Protein, and no carbohydrates or lipid were detected.
    Example 8. Lyophilized and canned microorganism Bordeteee a pertussis (strain Toma, phase 1), is cultivated (as in example 1), and then 10 l of the thus-obtained surface layer of the culture in liquid medium is passed through a column (column 5x2 cm , a flow rate of 60 ml / h with hydroxyapatite with subsequent washing with 300 ml of 0.01 M phosphate buffer (pH 7.0), after which 0.1 M phosphate buffer (pH 7.0) is passed through the column at a flow rate of 15 MP / hr
    The resulting active substance is condensed to 15 ml with the product Ficol-400, and then subjected to four times dialysis for a 24-hour period using 2 liters of distilled water, followed by four times dialysis for a total of 24 hours, using 1 l 0, 01 M phosphate buffer (pH 7.0) for equilibration.
    The biologically active substance is passed through a column with anti-BAO-antibody-Sepharose 4B (column with pa3MepaNM 1,8x13 cm), and then thoroughly washed with approximately 20 0.01 M phosphate buffer (pH 7.0), which contains 0, 1 M sodium chloride, followed by elution with 0.1 M glycine-hydrogen chloride (pH 3.0), which contains 2 mmol EDTA and 0.15 M sodium chloride, with a flow rate of 60 ml / h.
    The eluate is immediately neutralized with 1 M glycine buffer (pH 11.5).
    The active ingredient is collected and dialyzed three times for a total of 48 hours using 5 liters of distilled water to obtain 1.9 mg of white powder.
    The molecular weight of this substance is 70,000 ± 5,000 to obtain a single band with polyacrylamide gel electrophoresis (gel with a pH of 4.3). The product is characterized by the following chemical composition: 97 wt.% Or more protein, 1% or less carbohydrates, and no lipid was found. Example 9. Pharmacological action of the protein biologically active substance. Determination of the ability to accelerate insulin secretion. The ability to increase the rate of insulin secretion in a biologically active substance can be determined by the animal's response to insulin secretagogues of various types, and glucose is usually used as a stimulant for this purpose. Experimental animals. Males of Wistar rats (weighing from 13 to 140 g). Coarsely purified or purified biologically active substances of various strengths are dissolved in physiological saline and intravenous injections of 0.2 MP of each of these solutions under ether anesthesia into the femoral vein of an experimental rat, after which three days are measured for kg-1 substance. secretory insulin activity. 18-20 hours before the start of the experiment, rats are anchored. To measure the activity of plasma insulin concentration after glucose administration (µbed / mp) 1 / G (µbed / mg) Blood glucose concentration after glucose administration (mg / mp) The use of glucose in the blood to calculate the activity is due to the reason that the amount declined insulin is largely determined by blood sugar. The average value of the ratio l1 / ls for animals in whose body the experiment is administered. Unit of substance
    The average value of D1 / DKhZ for the control group
    The specific activity for each substance is obtained by dividing the unit by the amount of protein (Lowry method, etc.).
    The unit and dosage (in terms of protein) of the biologically active substance obtained in Example 1 are shown in Table. 6
    A sample of the purified biologically active substance shows a hypoid. the relationship between the reaction and the dosage, however, for the calculation in the unit and the purification stage for both the substance and its equivalent, the maximum possible linear segment was chosen.
    This substance allows you to accelerate
    insulin secretion, but it also has other pharmacologically beneficial properties, in particular, improved glucose tolerance, improved response, is resistant to insulin secretion, accelerated treatment
    diabetes caused by streptozotocin and improved glucose tolerance in hereditary diabetes. This effect lasts for several weeks to several months when the substance is administered to the body at the same time. This was observed in all experimental animals, including mice, rats and dogs, the pharmacological effect of such a substance not the tail vein of each rat was taken from 0.1 ml of blood, immediately after which a 30% glucose solution in an amount of 1 MP per X1 each 100 g was administered intrabranchically. body weight and exactly after 15 minutes after that, by analogy with the above, 0.1 ml of blood is again taken. The secretory insulin activity is determined by the difference in blood glucose and by the concentration of insulin in the blood before glucose administration and after glucose administration. The blood glucose concentration is measured according to the glucose oxidase method, and the insulin concentration is measured by the double antibody method. A parenteral solution for intravenous administration, used for a subsequent pharmacological experiment, is prepared in accordance with the above formulation. Method of calculating insulin secretion activity. Initially, the ratios l.1 / d6 of the active substance for the group of animals into which it is administered are obtained, and for the control group of animals, the following equation is obtained: Insulin concentration in plasma before administration of glucose (µed / ml) Blood glucose concentration before administration glucose unit (mg / mp) The unit for each active ingredient can be obtained using the following level: The average D1 / B ratio for animals in the control group varies to any significant degree in depending on the differences in animal species. The substance is used as an agent for treating diabetes. Currently, the treatment of diabetes depends on insulin injections or oral administration of antidiabetic drugs, however, the treatment is merely symptomatic, and (as it is considered at the present time) diabetes can be called an incurable disease. Moreover, the patient is forced to come to the hospital daily for insulin injection, and in these cases, the introduction of antidiabetic drugs is always associated with the danger of causing an abnormal deviation of blood glucose from the required level. The advantage of this substance is that it not only in itself has an extremely valuable action that causes insulin secretion, but also has an effect that causes insulin concentration in the blood to rise only when the glucose level in the blood rises under certain conditions (hyperglycemic especially when it enters the body, glucose, in particular, when eating), as a result of which the glucose content in the blood quickly returns from its elevated level to its normal level, moreover, its The action is maintained over a period of several weeks to months was waged after the same time of administration to an organism. Thus, in the case when the secretory insulin response to the level of glucose in the blood is rejected, the introduction of this substance into the body leads to a normal insulin secretion activity. Due to such properties, this substance is more widely used, i.e. It is not only useful as a medicine for the treatment of diabetes, its complications and age-related diseases caused by diabetes, but also has the potential to be used at the pre-diabetic stage of the disease as a prophylactic, therapeutic or diagnostic tool in the initial stage of diabetes for which there are still no drugs. The effect of accelerating insulin secretion of the proposed biologically active substance was tested on rats (Wishar strain Séishi rats) and dogs (both on the Sc1mc, and on the female dog PS7ROD dog or short-hound dog). When glucose, which is the most physiological actor, was administered to rats, a noticeable increase in insulin concentration in the blood was observed compared to what takes place in the control group of animals, regardless of the route of glucose administration to the body. A marked increase in reactivity was also noted with respect to hormone stimulants, in particular, glucogon (1 mg / kg) and epinephrine (200 µg / kg). An increase in the insulin secretion activity of tolbutamide (20 mg / kg) and glibenclamide (mg / kg), which are currently used as clinical antidiabetic agents, was also observed. On the basis of these results, it was found that the introduction into the body of the proposed substance significantly improves the reactivity of the body to ingestion of insulin secretagogues. The data on stimulation of reactivity with respect to various stimuli of insulin secretion in the body of beauty, which have previously been given a biologically active substance, are given in Table. 7. 1 μg of the substance is administered “nutrivenno” 3 days before the start of the experiment. The above table shows mean values and standard errors (SP) for 5 animals. Biologically active substances were administered intravenously to dogs and after 3 days to test the ability to stimulate insulin secretion, glucagon was injected into their bodies (by intravenous injection at a dosage of 25 μg / kg body weight). 18 hours before the start of the experiment, the experimental animals were injected. Experimental results for glucagon administration are given in Table 8. Acceleration, although insignificant / insulin secretion, was marked in comparison with control animals within 5 min after administration of glucagon at a dosage of 50 pg (in terms of protein); The same is true in all other cases, per kilogram of live weight, and the insulin secretion accelerated in proportion to the increase in dosage, reaching an almost maximum degree of reasch at a dosage of 1 µg / kg. The angshoic ability to enhance the extra-secretory secretion activity was noted when glucose was administered (through the mouth or by intravenous injection), as well as with epinephrine (see Table 9). These results show that with the administration of the substance in dogs, there is also a marked increase in reactivity with respect to insulin secretagogues. Data on the enhancement of insulin secretion after the introduction of glucagon into the body of dogs that have previously been given a biologically active substance BAS are given in Table. 8. Data on the enhancement of insulin recovery after the administration of glucose and epinephrine 5 minutes after the intravenous injection by injection into the body of dogs that have previously been given a BAS are given in Table. 9. Action for improving glucose tolerance. Glucose was administered orally to rats, and dogs were measured and glucose lowering and insulin concentrations in the blood of animals were measured to determine glucose tolerance. Glucose was given to rats at a dosage of 0.5 g / 100 g of live weight (crust), and to dogs at a dosage of 15 g / animal, with the animals preliminarily taking 18-20 hours before the experiment began. In rats and dogs that had previously been given a BAS, an increase in blood glucose concentration was markedly suppressed with a simultaneous increase in blood insulin, however, this insulin concentration quickly returned to the level at which glucose entered the body, in accordance with the normalization of glucose concentration in the blood, as a result of which, due to increased insulin secretion, no deviation of the glucose concentration in the blood from the normal level was noted (see Tables 10 and 11). These results demonstrated a marked improvement in glucose tolerance in animals that were injected with biologically active substances. Changes in the concentration of glucose in the blood and insulin in the plasma of dogs after glucose is introduced into their bodies are shown in Table. 10. Animals that were given BLV were administered intravenously at a dose of 1 μg (in terms of protein) BAS / kg for 3 days before the start of the experiment. Changes in the concentration of glucose in the blood and insulin in the plasma in the rat organism after glucose is given in Table. 11. Animals that received a BAS were injected intravenously at a dose of 0.5 μg of BAS 3 days before the start of this experiment. Prevention of diabetes, which is caused by streptozotocin, by the preliminary administration of biologically active substances. It is known that the administration of streptozotocin, hereinafter abbreviated as STZ, causes diabetes in experimental animals due to a noticeable destruction of the B cell of the islet of Langerhans. However, it was found that in rats that were injected with BAS, the STZ diabetes quickly healed, and Blood glucose and glucose tolerance returned to normal. The experiment was carried out according to this method, according to which 1 μg of BAS was administered to the rats first, and then, after 3-5 days, they were administered STZ (at a dosage of 5 mg / 100 g of live weight by intravenous injection). After 24 h after administration of STZ, hyperglycemia was observed in animals of both the control group and the group with which BAS was administered, however, in animals that were administered BAS, blood glucose levels gradually reached normal levels on the fifth and seventh days after administration of STZ and the concentration of insulin in the blood was also significantly higher than in animals of the control group (Table 12). Moreover, on the seventh day after the introduction of STZ, the experiment was repeated with the introduction of glucose, goat into the organism of animals and glucose tolerance was investigated (Table 13). In this case, when the rats were bound, the concentration of glucose in the blood became the same as for the control group (which was administered only STZ), and for the group that was administered biologically active substances; however, in the control group of animals, a clearly marked decrease in glucose was observed. tolerance compared with animals of the normal group. On the other hand, in animals that were injected with biologically active substances, the glucose tolerance was improved to such an extent that it could be compared with the glucose tolerance of animals of the normal group, and the insulin content in plasma in response to the introduction of glucose into the organism exceeded its normal animals. groups. Animals that had previously been given a BAS were recovered from diabetes, caused by the administered FCZ. in tab. 12 presents data on the prevention of diabetes caused by streptozotocin, prior to the introduction of biologically active substances. Data on the improvement of glucose tolerance in diabetes, which is caused by streptozotocin, prior to the introduction of biologically active substances, are given in Table. 13. Improvement of glucose tolerance through the introduction of biologically active substances in spontaneous diabetes in mice (or KK), similar to human diabetes. Conduct the following experiment. QC mice are used as experimental animals for such an experiment. Experiment pro-. water as follows. Glucose was administered through the mouth (at a dosage of 0.15 g / 20 g live weight) to mice aged 20 to 25 weeks, after which 5 animals were selected that demonstrate a certain lack of glucose tolerance compared to animals of the normal group (strain ddy) . As shown in the table. 14 in these cases, there was no decrease in the glucose content after its administration, and as a result, these animals could be considered as diabetic with certainty. Animals of this group (shk KK again injected through the mouth glucose on the third day after injection into the tail vein of the proposed substance. As a result, it was found that their glucose tolerance was significantly improved compared with what occurred before the introduction of the proposed substance. Glucose. the same tolerance of the mice is higher than that of the mice of the normal group. Thus, it can be considered that the administration of a BAS leads to a satisfactory therapeutic effect in the treatment of diabetes mellitus Data on the improvement of glucose tolerance in QL by introducing BAZ into their body are given in Table 1. The pharmacological activity of biologically active substances appears several hours after its introduction into the body, reaches a maximum level from 3 to 7 days, and then gradually The following are the results of experiments to determine the duration of action in the body of rats (0.5 µg) and dogs (1.0 µg / k. In dogs, studies were performed according to the definition of glucose tolerance in response to administration to orTable 1
    Casamic acid, g Yeast extract, g Primary acidic potassium phosphate, g Soluble starch, g
    0.5% solution of copper sulfate, ml
    1% calcium chloride solution, ml 4% magnesium chloride solution, MP Polypeptone, g 1% cystine solution,
    MP
    0.5% solution of iron sulfide, ml
    Sodium chloride, g
    2.5 glucose by intravenous injection on the 15th day after the administration of a BAS, as well as by determining the effect of insulin secretion in response to the administration of zinephrine and glucagon on the 29th and 42nd days (respectively) after the administration of the BAS. As a result, sufficient activity was observed on day 42 (Table 15). In rats, approximately 50% activity compared to the maximum activity (3 days after the administration of the BAS) was noted on the 28th day after the administration. Based on these results, it can be concluded that the pharmacological effect of BAS lasts for several weeks from several months, although the strength of this effect due to the dosage is uncertain. Data on the enhancement of insulin secretion in dogs, caused by the introduction of an intravenous injection of 25 µg / kg of gluCagon on day 42 after administration of the BAS, are given in Table. 15. Effective dose and route of administration. The effective dose of a BAS for treating a person is from 10 mg / kg of live weight to 1 µg / kg of live weight in the case of a purified sample of a substance; however, this substance can also be administered in doses up to 3 µg, which reach 2 µg / kg of live weight and even more. The most effective method of administration is intravenous injection, however, the proposed substance can also be administered in other ways. The proposed "1" method allows to obtain a biologically active substance that has the ability to enhance insulin secretion and improve glucose tolerance. f Continued table. one
    table 2
    Top layer of nutrient medium 10,000 2000 22,000 Hydrox and apitic column chromium 125 159 19.9 chromatography CM-Sepharous column 45 251 11.3 chromatography
    607 0.318 100 193.0 55 221.0 Note:
    lucosa
    Control group b ± 1
    Animals,
    received
    BAS
    Control group 8 ± 1
    Pinephrine animals that received BAS 18 ± b
    Note: mean value of mic / ml ± SP blood glucose, mg / mp: in the control group 125 ± 9,177 ± 14,123 ± 9 in animals administered 97 ± 3,117 ± 9,108 ± 7 Plasma insulin content, miced / ml: in the control 3 ± 2 17 ± 2 23 ± 15 groups in animals that were administered 9 ± 5 63 ± 10 1b ± b
    Table 8
    20i9
    7 ± 1
    89 ± 3b 8 + 1
    225 ± 28
     for 3 animals in appropriate cases.
    That blitz is 10 mean / ml for 3 animals, as appropriate. Table 9 18 ± 4 111 ± 19 113 ± 13 109 ± 1 18 ± 4 96 ± 6 88 ± 8 97 ± 3 8 ± 2 12 ± Z 8 ± з 8 ± 1 9 ± 1 lOil 20 ± 2 12 ± 4
    Concentration of glucose, in blood, mg / mp:
    control group b8 ± 1 102 ± 9
    animals receiving 51 ± 1 72 ± b y bash
    The content of insulin in plasma, ICED / MP:
    control group 19 ± 2
    animals, receiving 25 ± 4 126i9 second BAS
    Animals of the control group. The content of glucose in the blood MKED / ml 94 + 3 375 + 20 360 + 16 355 ± 8 351 ± 4
    Insulin content
    in plasma, miced / ml 20 ± 3 30 ± 3 26 ± 4
    Animals that have previously introduced a BAS
    Glucose content
    in blood, mg / ml 98 ± 2 358 ± 19 250 + 35 I99t41 145 ± 22
    Insulin content
    in plasma, mked / ml 69 ± 5 54 + 6
    Note: mean ± min dl for 5 animals
    as appropriate. Animals of the control group83 ± 7 14 ± 18 291 ± 27 (18 ± 2П22 ± 5) Animals, which. introduced BAV82 + 6 159 ± 16 177 ± 14 82 + 6 159 ± 16 (14 ± 2П49 + 2Г Animals normal 82 ± 3 l30tlJ 161 ± 5 t3, groups 7 + lf ((17 + 1G (42 ± 4Г
    Blood Glucose (mg / mp)
    Plasma Insulin Concentration (µbed / mL)
    Table 11
    108 + 4
    114 ± 4120 + 4
    48 ± 5
    b2 ± 669 ± 7
    47 ± 6
    41 ± 2
    41 + 555 ± 5
    b9 ± 7104 ± 18
    b1 ± b
    24 ± 2
    62 ± 4
    Table13 327 ± 23 264 ± 32 173 ± lO l4l ± 13 155 ± 4 138 ± 7 Animals of the normal group (ddy strain mice) Animals with diabetes (kishi of ddy strain) before injection into the body 117 ± b 300 ± 33 BAS
    After the introduction of BAS72 ± 3,150 ± 11,118 ± 9 into the body
    权利要求:
    Claims (1)
    [1]
    Claims of Invention A method for producing a biologically active substance capable of enhancing insulin secretion and improving glucose tolerance, which consists in the pathogenic strain Bordete Ea; pertussis is harvested into the feeder. Table 14 98 ± 7 214 ± 1b
    90 ± 8 63 ± 11
    . Spreadsheets
    A medium containing a source of carbon, nitrogen, mineral salts and growth stimulants, followed by separation of the protein fraction with a molecular weight of 77,000 ± 6,400, determined by gel-filtration and purification of the target product, from the culture fluid. 339 ± 13,192 ± 9,144 ± 14 Za8 ± 21 32b ± 40 310 ± 39
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    同族专利:
    公开号 | 公开日
    JPS5635154B2|1981-08-14|
    ES467067A1|1978-11-01|
    IE780205L|1978-08-01|
    DK152058B|1988-01-25|
    GB1569046A|1980-06-11|
    DK45378A|1978-08-02|
    CH640244A5|1983-12-30|
    CA1105863A|1981-07-28|
    NL7801137A|1978-08-03|
    NZ186361A|1980-10-08|
    FR2378793B1|1982-10-22|
    AU513963B2|1981-01-15|
    CS216547B2|1982-11-26|
    HU179497B|1982-10-28|
    DE2803397A1|1978-08-03|
    AU3285478A|1979-08-09|
    FR2378793A1|1978-08-25|
    US5000953A|1991-03-19|
    ZA7855B|1978-11-29|
    DE2803397C2|1990-01-25|
    IE46649B1|1983-08-10|
    SE7801106L|1978-08-02|
    JPS5396392A|1978-08-23|
    BE863477A|1978-07-31|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    RU2281094C2|1993-09-15|2006-08-10|Санкио Компани, Лимитед|Method for treatment of impaired glucose tolerance|
    JPS59206316A|1983-05-11|1984-11-22|Kaken Pharmaceut Co Ltd|Active substance derivative for enhancing secretion of insulin and production thereof|
    US6143786A|1999-02-02|2000-11-07|Novartis Nutrition Ag|Oral arginine and insulin secretion|
    WO2001085256A2|2000-05-05|2001-11-15|Novo Nordisk A/S|Critical illness neuropathy|
    US6986882B2|2002-01-17|2006-01-17|Astrazeneca Ab|Therapy for functional dyspepsia|
    US20030199445A1|2002-02-07|2003-10-23|Knudsen Lotte Bjerre|Use of GLP-1 compound for treatment of critically ill patients|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP1039777A|JPS5635154B2|1977-02-01|1977-02-01|
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