CN114569707A - Application of adenosine deaminase or modified substance thereof in preparation of diabetes treatment drug - Google Patents

Application of adenosine deaminase or modified substance thereof in preparation of diabetes treatment drug Download PDF

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CN114569707A
CN114569707A CN202011372874.1A CN202011372874A CN114569707A CN 114569707 A CN114569707 A CN 114569707A CN 202011372874 A CN202011372874 A CN 202011372874A CN 114569707 A CN114569707 A CN 114569707A
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adenosine deaminase
diabetes
mouse
gluconeogenesis
blood glucose
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CN114569707B (en
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张建法
葛文浩
杨云霞
赵扬
丁昭
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Nanjing University of Science and Technology
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Nanjing University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04004—Adenosine deaminase (3.5.4.4)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses application of adenosine deaminase or a modifier thereof in preparing a medicament for treating diabetes. According to the invention, after the diabetes mouse is treated by the naturally extracted adenosine deaminase, the recombinant adenosine deaminase or the polyethylene glycol modifier thereof, the tolerance of the mouse to pyruvic acid is relieved, the blood sugar of the diabetes mouse can be effectively reduced, and the gluconeogenesis level of the mouse is improved. The invention discovers for the first time that the adenosine deaminase or the modified substance thereof has obvious inhibition effect on gluconeogenesis of type 2 diabetes, and the adenosine deaminase and the modified substance thereof have potential application prospect in preparing the medicament for treating type 2 diabetes.

Description

Application of adenosine deaminase or modified substance thereof in preparation of diabetes treatment drug
Technical Field
The invention belongs to the technical field of biological medicines, and relates to application of adenosine deaminase or a modifier thereof in preparation of a diabetes treatment medicine.
Background
Diabetes is a common metabolic endocrine disease, has no symptoms in the early clinical stage, and has the symptoms of polyphagia, polydipsia, polyuria, polydipsia, hunger, emaciation or obesity, fatigue, weakness and the like until the symptom stage. According to world health organization data, type 2 diabetes has become a global epidemic affecting more than 4 hundred million people in 2014. More than 90% of diabetes mellitus is type 2 diabetes mellitus, mainly caused by insulin resistance and relative insulin hyposecretion. Type 2 diabetics do not rely on exogenous insulin for survival, but some require insulin to control hyperglycemia.
Uncontrolled increases in blood glucose concentration are hallmarks of type 2 diabetes, and prolonged increases in blood glucose can lead to serious complications, including cardiovascular complications, neuropathy, renal failure, and cancer. It is widely believed that hyperglycosemia is the leading cause of elevated blood glucose levels in type 2 diabetic patients. Therefore, reducing hepatic gluconeogenesis is a viable strategy for managing blood glucose levels in type 2 diabetes. Evidence shows that hepatic gluconeogenesis abnormality is closely related to diseases such as obesity, non-alcoholic fatty liver, diabetes and the like. Inhibiting gluconeogenesis can be used for treating obesity, non-alcoholic fatty liver disease, and diabetes.
Adenosine Deaminase (ADA) is a purine catabolic enzyme that converts adenosine to inosine, thereby helping to reduce the levels of adenosine present in tissues and cells. Adenosine deaminase deficiency is a rare inherited purine metabolic disease characterized by immunodeficiency, dysplasia and metabolic abnormalities. Lack of ADA leads to accumulation of toxic metabolites, causing cell-mediated humoral immunodeficiency, resulting in Severe Combined Immunodeficiency (SCID). Hematopoietic stem cell transplantation can cure combined immunodeficiency, but is often limited by good donor matching. Other treatments include Enzyme Replacement Therapy (ERT) with bovine ethylene glycol enzyme (PEG-ADA), and more recently gene therapy. PEG-ADA has been used in numerous patients worldwide, which can restore the metabolic detoxification function and protect the immune function of patients, and keep the patients in a clinically good state. However, there is no report on the use of adenosine deaminase or its modification for inhibiting hepatic gluconeogenesis.
Disclosure of Invention
The invention aims to provide application of adenosine deaminase or a modifier thereof in preparing a medicament for treating diabetes.
The diabetes mellitus is type 2 diabetes mellitus.
The adenosine deaminase is a protein for metabolizing adenosine, and comprises adenosine deaminase extracted from animal tissues, adenosine deaminase recombined with other genes, and the like.
In the specific embodiment of the invention, the adenosine deaminase is a naturally extracted bovine adenosine deaminase or a murine adenosine deaminase expressed by escherichia coli, and the nucleotide sequence of the adenosine deaminase is shown as SEQ ID No. 1.
The modified adenosine deaminase is obtained by chemically modifying adenosine deaminase, so that the stability is improved, and the half-life period is prolonged.
The invention also provides a diabetes treatment drug which contains one or more of adenosine deaminase or a modifier thereof. Furthermore, the diabetes treatment medicine also contains a pharmaceutically acceptable carrier or excipient and is prepared into a pharmaceutically acceptable dosage form.
Compared with the prior art, the invention has the following remarkable advantages: (1) adenosine deaminase is a protein naturally possessed by organisms, is wide in source and has excellent biocompatibility; (2) it is disclosed for the first time that adenosine deaminase and its modifier can produce obvious inhibiting effect on gluconeogenesis.
Drawings
FIG. 1 is a graph showing the effect of naturally extracted bovine adenosine deaminase on fasting blood glucose in diabetic mice.
FIG. 2 is a graph showing the effect of natural bovine adenosine deaminase on pyruvate tolerance in diabetic mice.
FIG. 3 is a graph showing the effect of PEG-modified naturally-extracted bovine adenosine deaminase on fasting blood glucose in diabetic mice.
FIG. 4 is a graph showing the effect of polyethylene glycol modified naturally extracted bovine adenosine deaminase on pyruvate tolerance in diabetic mice.
FIG. 5 is a graph showing the effect of E.coli expressing mouse adenosine deaminase on fasting plasma glucose in diabetic mice.
FIG. 6 is a graph showing the effect of E.coli expression of mouse adenosine deaminase on pyruvate tolerance in diabetic mice.
FIG. 7 is a graph showing the effect of PEG-modified Escherichia coli expressing mouse adenosine deaminase on fasting blood glucose in diabetes.
FIG. 8 is a graph showing the effect of polyethylene glycol modified Escherichia coli expressing mouse adenosine deaminase on pyruvate tolerance in diabetic mice.
Detailed Description
The present invention will be described in more detail with reference to the following examples and the accompanying drawings. The methods used in the following examples are those conventionally used in the art unless otherwise specified.
The raw materials used in the following examples are all commercially available products unless otherwise specified.
The following examples relate to adenosine deaminase, including by purchase or self-made by the applicant. The adopted natural extracted bovine adenosine deaminase is purchased from Shanghai biological engineering Limited company and self-made rat adenosine deaminase expressed by escherichia coli.
The following examples relate to adenosine deaminase modifications that include those made by the applicant or purchased as such. Modifying naturally extracted bovine adenosine deaminase by polyethylene glycol (purchased from Beijing Kanjin pharmaceutical technology Co., Ltd.), and modifying murine adenosine deaminase expressed by Escherichia coli by polyethylene glycol.
Example 1 effect of natural extraction of bovine adenosine deaminase on gluconeogenesis in diabetes.
(1) Experiment grouping
SPF-grade male db/db mice were selected, bred for 8 weeks under standard experimental conditions: 12 hours light-12 hours dark cycle, free intake of water and food. Two groups were randomized, control and adenosine deaminase treated. The control group was treated with the corresponding drug medium (PBS buffer); adenosine deaminase treatment group, injecting natural extracted bovine adenosine deaminase (5U/g) into abdominal cavity.
(2) Fasting blood glucose determination
After 24 hours of intraperitoneal injection of adenosine deaminase, 3 per group are taken for determining gluconeogenesis rate, and the specific method comprises the following steps: after an overnight fast, the fasting blood glucose was measured by tail vein blood sampling and recorded as fasting blood glucose of the mice.
(3) Pyruvic acid tolerance test
After injecting adenosine deaminase into the abdominal cavity for 24 hours, taking 3 capsules each group for measuring gluconeogenesis rate, wherein the specific method comprises the following steps: after fasting for 16 hours, tail vein blood sampling is carried out to measure fasting blood glucose, and the blood glucose value is recorded as 0 minute; thereafter, sodium pyruvate was intraperitoneally injected at a dose of 2g/kg body weight, and blood glucose was measured by tail vein blood sampling for 15,30,60, and 120 minutes, respectively. Thereby detecting gluconeogenesis levels.
(4) Results of the experiment
As shown in FIG. 1, the results of the experiment show that the fasting blood sugar of the diabetic mice is obviously reduced after the treatment of adenosine deaminase. The experimental results combined with figure 2 show that the pyruvic acid tolerance of the mice is relieved after the diabetic mice are treated by the adenosine deaminase. The results show that the natural extraction of the bovine adenosine deaminase can effectively reduce the blood sugar of a diabetic db/db mouse and improve the gluconeogenesis level of the mouse.
Example 2 Effect of polyethylene glycol-modified Natural extract of bovine adenosine deaminase on gluconeogenesis in diabetic mice
(1) Preparation of polyethylene glycol modified natural extracted bovine adenosine deaminase
ADA was diluted to 500U/ml with 1ml sterile PBS (10mmol/l, pH 9.0). Then, mPEG-SPA (Beijing Kanjin combination pharmaceutical technology Co., Ltd.) having a molecular weight of 20kDa was added thereto at a final concentration of 100mg/ml, and the mixture was mixed at room temperature for 5 hours. Finally, PBS (10mmol/l, pH7.4) was added and the PEG-ADA was diluted to a final concentration of 150U/ml.
(2) Experiment grouping
SPF-grade male db/db mice were selected, bred for 8 weeks under standard experimental conditions: 12 hours light-12 hours dark cycle, free intake of water and food. Two groups were randomized, control and adenosine deaminase treated. The control group was treated with the corresponding drug vehicle (PBS); adenosine deaminase treatment group, i.e. injecting polyethylene glycol modified natural extracted bovine adenosine deaminase (1.5U/g) into abdominal cavity.
(3) Fasting blood glucose determination
After injecting adenosine deaminase into the abdominal cavity for 24 hours, taking 3 capsules each group for measuring gluconeogenesis rate, wherein the specific method comprises the following steps: after overnight fasting, tail vein blood was collected to determine fasting blood glucose, which is recorded as fasting blood glucose of mice.
(4) Pyruvic acid tolerance test
After injecting adenosine deaminase into the abdominal cavity for 24 hours, taking 3 capsules each group for measuring gluconeogenesis rate, wherein the specific method comprises the following steps: after fasting for 16 hours, tail vein blood sampling is carried out to measure fasting blood glucose, and the blood glucose value is recorded as 0 minute; thereafter, sodium pyruvate was intraperitoneally injected at a dose of 2g/kg body weight, and blood glucose was measured by tail vein blood sampling for 15,30,60, and 120 minutes, respectively. Thereby detecting gluconeogenesis levels.
(5) Results of the experiment
Referring to FIG. 3, the results of the experiment show that the fasting blood glucose of the diabetic mice is significantly reduced after the treatment with adenosine deaminase. The experimental results combined with figure 4 show that the pyruvate tolerance of the mice is relieved after the diabetes mice are treated by adenosine deaminase. The results show that the polyethylene glycol modified naturally extracted bovine adenosine deaminase can effectively reduce the blood sugar of a diabetic db/db mouse and improve the gluconeogenesis level of the mouse.
EXAMPLE 3 Effect of Escherichia coli-expressed murine adenosine deaminase on gluconeogenesis in diabetic mice
(1) Preparation of mouse adenosine deaminase expressed by Escherichia coli
The mouse adenosine deaminase fragment DNA (SEQ ID NO.1) was cloned into the vector pGE-x-4T-1 by PCR amplification. The resulting plasmid was transformed into E.coli BL21(DE 3). Ada transformed E.coli was cultured in LB medium supplemented with 100mol/l ampicillin (see section pGE-x-4T-1). 2mL of the overnight culture was transferred to 200mL of fresh LB medium and cultured at 37 ℃ under a shaking table rotation speed of 230rpm for about 2 hours until OD of the bacteria-containing medium600Up to 0.6. Filter-sterilized isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2mmol/l and incubation was continued at 28 ℃ for about 24 hours with a shaker rotation speed of 2300 rpm. The cells were collected by centrifugation at 4 ℃ at 13000rpmResuspended in 25ml of Phosphate Buffered Saline (PBS) lysis buffer supplemented with 10mm protease inhibitor and sonicated (power: 300W, working time 5 seconds, pause time 10 seconds, total number of 30). The GST-tagged protein was purified by GST affinity chromatography. GST-ADA was first purified on glutathione sepharose 4B affinity column, and then GST tag was removed overnight at thrombin 4 ℃. Thrombin was removed by benzamidine-agarose chromatography immediately after incubation.
(2) SPF-grade male db/db mice were selected and bred for 8 weeks under standard experimental conditions: 12 hours light-12 hours dark cycle, free intake of water and food. Two groups were randomized, control and adenosine deaminase treated. The control group was treated with the corresponding drug vehicle (PBS); in the adenosine deaminase treatment group, mouse adenosine deaminase (5U/g) was injected intraperitoneally.
(3) Fasting blood glucose determination
After injecting adenosine deaminase into the abdominal cavity for 24 hours, taking 3 capsules each group for measuring gluconeogenesis rate, wherein the specific method comprises the following steps: after an overnight fast, the fasting blood glucose was measured by tail vein blood sampling and recorded as fasting blood glucose of the mice.
(4) Pyruvic acid tolerance test
After injecting adenosine deaminase into the abdominal cavity for 24 hours, taking 3 capsules each group for measuring gluconeogenesis rate, wherein the specific method comprises the following steps: after fasting for 16 hours, tail vein blood sampling is carried out to measure fasting blood glucose, and the blood glucose value is recorded as 0 minute; thereafter, sodium pyruvate was intraperitoneally injected at a dose of 2g/kg body weight, and blood glucose was measured by tail vein blood sampling for 15,30,60, and 120 minutes, respectively. Thereby detecting gluconeogenesis levels.
(5) Results of the experiment
Referring to FIG. 5, the results of the experiment show that the fasting blood glucose of the diabetic mice is significantly reduced after the treatment with adenosine deaminase. The experimental results combined with FIG. 6 show that the pyruvate tolerance of the mice is relieved after the mice are treated by adenosine deaminase. The results show that the mouse adenosine deaminase can effectively reduce the blood sugar of db/db mice with diabetes and improve the gluconeogenesis level of the mice.
EXAMPLE 4 Effect of polyethylene glycol-modified E.coli-expressed murine adenosine deaminase on gluconeogenesis in diabetic mice preparation of E.coli-expressed murine adenosine deaminase
The mouse adenosine deaminase fragment DNA (SEQ ID NO.1) was cloned into the vector pGE-x-4T-1 by PCR amplification. The resulting plasmid was transformed into E.coli BL21(DE 3). Ada transformed E.coli was cultured in LB medium supplemented with 100mol/l ampicillin (see section pGE-x-4T-1). 2mL of the overnight culture was transferred to 200mL of fresh LB medium and cultured at 37 ℃ under a shaking table rotation speed of 230rpm for about 2 hours until OD of the bacteria-containing medium600Up to 0.6. Filter-sterilized isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2mmol/l and incubation was continued at 28 ℃ for about 24 hours with a shaker rotation speed of 2300 rpm. The cells were collected by centrifugation (13000rpm) at 4 ℃ and resuspended in 25ml of Phosphate Buffered Saline (PBS) lysis buffer to which 10mm protease inhibitor was added, and sonicated (power: 300W, working time 5 seconds, pause time 10 seconds, total number of 30). The GST-tagged protein was purified by GST affinity chromatography. GST-ADA was first purified on glutathione sepharose 4B affinity column, and then GST tag was removed overnight at thrombin 4 ℃. Thrombin was removed by benzamidine-agarose chromatography immediately after incubation.
(2) Preparation of polyethylene glycol modified mouse adenosine deaminase
ADA was diluted to 500U/ml with 1ml sterile PBS (10mmol/l, pH9.0). Then, mPEG-SPA (Beijing Kanjin combination pharmaceutical technology Co., Ltd.) having a molecular weight of 20kDa was added thereto at a final concentration of 100mg/ml, and the mixture was mixed at room temperature for 5 hours. Finally, PBS (10mmol/l, pH7.4) was added and the PEG-ADA was diluted to a final concentration of 150U/ml.
(3) Experiment grouping
SPF-grade male db/db mice were selected, bred for 8 weeks under standard experimental conditions: 12 hours light-12 hours dark cycle, free intake of water and food. Two groups were randomized, control and adenosine deaminase treated. The control group was treated with the corresponding drug vehicle (PBS); in the adenosine deaminase treatment group, polyethylene glycol modified mouse adenosine deaminase (1.5U/g) was injected intraperitoneally.
(4) Fasting blood glucose determination
After injecting adenosine deaminase into the abdominal cavity for 24 hours, taking 3 capsules each group for measuring gluconeogenesis rate, wherein the specific method comprises the following steps: after an overnight fast, the fasting blood glucose was measured by tail vein blood sampling and recorded as fasting blood glucose of the mice.
(5) Pyruvic acid tolerance test
After injecting adenosine deaminase into the abdominal cavity for 24 hours, taking 3 capsules each group for measuring gluconeogenesis rate, wherein the specific method comprises the following steps: after fasting for 16 hours, tail vein blood sampling is carried out to measure fasting blood glucose, and the blood glucose value is recorded as 0 minute; thereafter, sodium pyruvate was intraperitoneally injected at a dose of 2g/kg body weight, and blood glucose was measured by tail vein blood sampling for 15,30,60, and 120 minutes, respectively. Thereby detecting gluconeogenesis levels.
(6) Results of the experiment
Referring to FIG. 7, the results of the experiment show that fasting blood glucose of diabetic mice is significantly reduced after the mice are treated with adenosine deaminase. The results of the experiment combined with FIG. 8 show that the pyruvate tolerance of the mice is relieved after the mice with diabetes are treated by adenosine deaminase. The results show that the polyethylene glycol modified mouse adenosine deaminase can effectively reduce the blood sugar of a diabetic db/db mouse and improve the gluconeogenesis level of the mouse.
Sequence listing
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Application of <120> adenosine deaminase or modifier thereof in preparation of diabetes treatment drugs
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Claims (8)

1. Application of adenosine deaminase or its modifier in preparing medicine for treating diabetes is disclosed.
2. The use according to claim 1, wherein the diabetes is type 2 diabetes.
3. The use of claim 1, wherein the adenosine deaminase is an extracted from animal tissue or a genetically modified adenosine deaminase.
4. The use of claim 1, wherein the adenosine deaminase is a naturally-extracted bovine adenosine deaminase or a murine adenosine deaminase expressed by escherichia coli, and the nucleotide sequence of the adenosine deaminase is shown as SEQ ID No. 1.
5. The use of claim 1, wherein the adenosine deaminase modification compound is an adenosine deaminase that has been chemically modified to increase stability and half-life.
6. The use of claim 5, wherein the chemical modification includes but is not limited to polyethylene glycol modification, dextran modification or heparin modification.
7. A medicine for treating diabetes contains adenosine deaminase or one or more of its modified substances.
8. The diabetes treatment drug according to claim 7, characterized in that the diabetes treatment drug further comprises a pharmaceutically acceptable carrier or excipient, and is prepared into a pharmaceutically acceptable dosage form.
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Citations (3)

* Cited by examiner, † Cited by third party
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