WO2018171535A1 - Protéine de fusion de proinsuline recombinante humaine, son procédé de préparation et son utilisation - Google Patents

Protéine de fusion de proinsuline recombinante humaine, son procédé de préparation et son utilisation Download PDF

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WO2018171535A1
WO2018171535A1 PCT/CN2018/079375 CN2018079375W WO2018171535A1 WO 2018171535 A1 WO2018171535 A1 WO 2018171535A1 CN 2018079375 W CN2018079375 W CN 2018079375W WO 2018171535 A1 WO2018171535 A1 WO 2018171535A1
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fusion protein
composition
human proinsulin
recombinant human
proinsulin fusion
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PCT/CN2018/079375
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English (en)
Chinese (zh)
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刘晋宇
郝德顺
刘菲琳
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吉林大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present invention relates to a recombinant protein, and more particularly to a recombinant human proinsulin fusion protein and use thereof in the preparation of a hypoglycemic agent.
  • Diabetes is a group of clinical syndromes caused by defects in insulin secretion and/or function, characterized by elevated chronic blood glucose. Diabetes is a metabolic syndrome associated with many factors such as heredity and environment. It is characterized by metabolic disorders of carbohydrates, fats, and proteins. Chronic diabetes often leads to complications of one or more organs, such as diabetic nephropathy, peripheral vascular disease of the lower extremities, and diabetic foot. These organ dysfunctions and failures are the leading causes of death and disability in diabetes.
  • Insulin is a hormonal drug used by diabetics to control blood sugar levels. It is widely used in patients with type I and type II diabetes to replace or supplement the deficiency of insulin secretion in the body.
  • insulin drugs for clinical use are subcutaneous injection forms. Insulin injection often causes inconvenience and pain to the patient, and there are many adverse reactions, such as pain at the injection site, bleeding, subcutaneous nodule formation and the like. Insulin injection may lead to hyperinsulinemia, and severe cases of hypoglycemia, coma and even shock.
  • allergic reactions may occur after injection, and even anaphylactic shock may occur in severe cases. Therefore, it is extremely important to develop an insulin drug that does not require subcutaneous injection.
  • Insulin is specifically expressed and secreted by islet ⁇ cells, first expressed as pro-proinsulin, and the signal peptide is cleaved off in the endoplasmic reticulum and folded to form a secondary structure, which is transported to the Golgi apparatus. Under the action of Golgi endonuclease PC2 and PC1/3, the C peptide was cleaved to make it mature insulin. Since PC2 and PC1/3 are only expressed in islet ⁇ cells, other tissue cells cannot process proinsulin due to lack of endonuclease PC2 and PC1/3.
  • Cell-penetrating peptide is a kind of functional polypeptide that can efficiently pass the biofilm barrier. It is generally less than 20 amino acid residues. According to biochemical properties, it is divided into two types: cationic membrane peptides and amphiphilic peptides. . Common cell-penetrating peptides include TAT, penetratin, oligoarginine and the like. The membrane-penetrating peptide and the biomacromolecule are linked by chemical synthesis or construct a fusion-expressed protein, which can effectively promote transmembrane absorption of biomacromolecules.
  • the Chinese invention patent ZL200410039129.X provides a expression and application of a transduction peptide-human proinsulin fusion protein, but its embodiment does not show that the technical scheme can prepare a biologically active transduction peptide-insulin. The result of the original fusion protein. The reason may be as follows: 1.
  • the expression host used for the recombinant plasmid is clone competent cells JM109 and DH5 ⁇ .
  • Example 4 did not match its corresponding Figure 3:
  • Example 4 is temperature-induced expression, and Figure 4 In the description of 3, IPTG induced expression, and the SDS-PAGE electrophoresis pattern of Fig. 3 could not reveal the expression product band, and the electropherogram was "blackboard”.
  • the density scan described in Example 4 could not be performed and the expression product was calculated.
  • the ratio of the nucleotide sequence and the amino acid sequence of the fusion protein is inconsistent with the primary structure of the fusion protein of Figure 1.
  • the transduction peptide TAT is located at the amino terminus of the fusion protein, while the transduction peptide is located in Figure 1.
  • Carboxy end. 4 Example 4 describes that the fusion protein exists as an inclusion body, however, the "beneficial effect" portion and the purified portion of Example 7 do not have any step of renaturation of the fusion protein, and thus it is impossible to obtain an active fusion protein.
  • the expression vector used is a common E. coli expression plasmid, but in the technical scheme, the vector can be expressed by transfecting yeast cells, insect cells and mammalian cells, and no relevant experimental evidence is provided; 6 no protein organism is provided in the patent. Learn the activity test results.
  • Another object of the present invention is to provide a method for producing a recombinant human proinsulin fusion protein to obtain a polypeptide or protein having biological activity involved in a sugar metabolism process.
  • Still another object of the present invention is to provide a use of a recombinant human proinsulin fusion protein for the preparation of a medicament for the treatment and prevention of disorders associated with glucose metabolism.
  • Still another object of the present invention is to provide a use of a recombinant human proinsulin fusion protein for the preparation of a medicament for the treatment and prevention of diabetes.
  • composition referred to in the present invention further includes various pharmaceutical excipients compatible with the compound or composition contained therein to prepare a dosage form suitable for drug delivery, such as, but not limited to, aqueous solution pills, powders, tablets.
  • excipients may be conventionally used in various preparations such as, but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrators, and lubricants; It may also be selected for use in accordance with the substance, such as emulsifiers, solubilizers, bacteriostatic agents, analgesics and antioxidants, etc., such excipients can effectively improve the stability of the compounds contained in the composition and Solubility or change the release rate and absorption rate of the compound, etc., thereby improving the metabolism of various compounds in the living body, thereby enhancing the administration effect of the composition.
  • excipients such as, but not limited to, gelatin, albumin, chitosan, etc.
  • excipients such as, but not limited to, gelatin, albumin, chitosan, etc.
  • Polyether and polyester polymer materials such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate and copolymers thereof.
  • the main manifestations of "favorable administration" are: but not limited to improving the therapeutic effect, improving bioavailability, reducing toxic side effects, and improving patient compliance.
  • excipients In aqueous solution injections, excipients generally include isotonic agents and buffers, as well as the necessary emulsifiers (eg, Tweeen-80, Pluronic, and Poloxamer, etc.), solubilizers, and bacteriostats. In addition, it also includes other pharmaceutically acceptable excipients such as antioxidants, pH adjusters and analgesics.
  • Excipients for the preparation of oral liquid preparations generally include solvents, as well as the necessary flavoring, bacteriostatic, emulsifying, and coloring agents.
  • Excipients for preparing tablets generally include fillers (eg, starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, mannitol, etc.), binders (eg: ethanol, starch slurry, sodium carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, gelatin solution, sucrose solution and aqueous solution of polyvinylpyrrolidone Or alcohol solution, etc.), disintegrants (such as: dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone and croscarmellose sodium) and lubricants (such as: Magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, magnesium lauryl sul
  • Excipients used in the preparation of emulsions are generally water, oils (e.g., fatty acids), emulsifiers, and necessary preservatives and flavoring agents.
  • the excipients used to make the granules are similar to tablets, but the granulation process is different. If necessary, the prepared granules are mixed with a glidant and then filled into capsules to obtain a capsule.
  • organism refers to humans, wild animals and livestock (Livestock).
  • Wild animals are animals that have not been artificially domesticated in their natural state.
  • Livestock are animals that are artificially raised to provide a source of food, such as, but not limited to, dogs, cats, rats, rats, hamsters, pigs, rabbits, cows, buffalo, bulls, sheep, goats, geese, and chickens.
  • the "patient” or “organism” to which the treatment is administered preferentially selects a mammal, especially a human.
  • prevention refers to various means or measures for preventing the occurrence or development of a disease, including medical, physical or chemical means, before and after the disease is not recognized by clinical standards, to prevent and reduce various diseases.
  • the occurrence or development of symptoms refers to various means or measures for preventing the occurrence or development of a disease, including medical, physical or chemical means, before and after the disease is not recognized by clinical standards, to prevent and reduce various diseases. The occurrence or development of symptoms.
  • prevention of diabetes means that the composition of the present invention is used for a clinical patient who has not yet met the "diabetes” clinical index, and will gradually develop into a potential patient clinically defined as “diabetes” over time. Improve the tolerance of these patients to glucose, promote the body's ability to glucose metabolism, and increase the body's sensitivity to insulin. Such potential patients often suffer from "Metabolic Syndrome” (Annnu. Rev. Nutri., 2005, 25, 391-406; Annnu. Rev. Med., 2005, 56, 45-62; Nat. Rev. Drug. Disc., 2006, 5, 295-309; Nat. Rev.
  • treatment means to prevent or reduce the occurrence or development of a disease, and to inhibit, contain, alleviate, ameliorate, slow down, stop, delay or reverse the progression or aggravation of the disease course. / or various indicators of disease, disorder, or pathological state at the time of administration include alleviating or reducing symptoms or complications, or curing or eliminating a disease, disorder, or condition.
  • treating diabetes means that the composition of the present invention is used for clinical diagnosis of "diabetes", improving tolerance of glucose to these patients, promoting the body's ability to glucose metabolism, and increasing the sensitivity of the body to insulin. Sex. In turn, the patient's postprandial and fasting blood glucose is controlled to a normal level. As the ability to glucose metabolism is increased, the occurrence and development of various cardiovascular diseases, chronic renal failure, retinopathy, neuropathy and microangiopathy caused by long-term hyperglycemia are alleviated.
  • food is meant to include the individual compounds, compositions or extracts provided herein that are made into a single compound or composition that is edible.
  • the production and manufacture of such a single compound or composition should be in accordance with relevant food safety standards, but such food safety standards must not limit the invention.
  • health product means that a single compound or composition comprising the various compounds, compositions or extracts provided by the present invention is made into an edible compound for administration to a patient for preventing and treating diseases. purpose. It belongs to the food referred to in the present invention, but its production, manufacture and sale should also comply with various relevant requirements, standards and specifications.
  • drug means a single compound which can be used for preventing or treating a certain disease, a composition formed of a plurality of compounds, a Chinese medicinal material and an extract thereof, or a composition containing a single compound as a main active ingredient. Or formulation, also refers to a composition or formulation comprising a plurality of compounds as active ingredients. “Drug” should be understood to refer not only to the products approved and approved for production by the administrative agencies established by the laws of a country, but also to the inclusion of a single compound as an active ingredient in order to obtain approval and approval of production. Various forms of matter. "Formation” is understood to mean obtained by chemical synthesis, biotransformation or purchase.
  • the recombinant human proinsulin fusion protein provided by the invention has a furin enzyme recognition site at the junction of the human proinsulin C peptide with the B chain, the C peptide and the A chain, and the 10th amino acid of the B chain is replaced by histidine. Aspartic acid.
  • the recombinant human proinsulin fusion protein of the present invention can penetrate the epidermal cell membrane, and the recombinant human proinsulin fusion protein is processed into mature insulin by the epithelial cells. Therefore, it can be administered by transdermal administration, as well as oral mucosal administration (eg, oral patch), tongue mucosa or sublingual administration (eg, buccal tablets), respiratory mucosal administration (eg, spray), vaginal mucosa (eg, suppositories) and ocular administration (eg, eye drops) to improve the patient's glucose metabolism, especially in diabetic patients.
  • oral mucosal administration eg, oral patch
  • tongue mucosa or sublingual administration eg, buccal tablets
  • respiratory mucosal administration eg, spray
  • vaginal mucosa eg, suppositories
  • ocular administration eg, eye drops
  • a composition comprising the protein of SEQ ID No 2 to administer a recombinant human proinsulin fusion protein to an organism via epithelial cells and mucosal cells.
  • a method for producing a recombinant human proinsulin fusion protein which is constructed by a vector as shown in SEQ ID No. 1, constructed, isolated, purified and purified to obtain a protein having the sequence of SEQ ID No. 2.
  • prokaryotic expression system of recombinant human proinsulin fusion protein is as follows:
  • Escherichia coli was used as an expression vector and cultured overnight in LB medium.
  • the overnight culture was cultured in a fresh LB medium on the next day at a ratio of 1:1.5 to 100, and fermentation was continued for 1 hour to 24 hours.
  • the E. coli culture was disrupted by sonication, and the recombinant protein was dissolved in a sterilizing buffer containing a high concentration of a denaturant (8 mol/L urea or 6 mol/L guanidine hydrochloride).
  • the ultrasonic cleavage product was filtered at 0.45 ⁇ m to remove particulate matter, and injected into a nickel chelate agarose column to wash off the heteroprotein, and eluted with a gradient of 1.0-1000.0 mM imidazole to collect a sample of the A280 nm elution peak.
  • the recombinant human proinsulin fusion protein was diluted in a refolding buffer by a pulse method at a ratio of 1:100.
  • the recombinant human proinsulin fusion protein provided by the invention has a nucleotide sequence similarity of only 38.81% and an amino acid sequence similarity of 61.87% compared with the prior art, and provides detailed technical solutions, implementation methods and The experimental results indicate that a biologically active recombinant human proinsulin fusion protein has been obtained.
  • the recombinant human proinsulin fusion protein provided by the invention has better stability, can pass through various biofilm barriers efficiently, has higher drug absorption rate, can be percutaneously absorbed, sublingually, and transmucosa (oral cavity). Absorption of mucous membranes, tongue mucosa, nasal mucosa, and absorption through the respiratory mucosa and vaginal mucosa. Each organ promotes different insulin absorption patterns and has different absorption effects.
  • the present invention obtains a high concentration of a biologically active recombinant human proinsulin fusion protein by a simple and convenient preparation method.
  • Figure 1 is a prokaryotic expression vector map of the recombinant human proinsulin fusion protein of the present invention
  • FIG. 2 is a flow chart of a method for purifying and renaturation of a recombinant human proinsulin fusion protein of the present invention
  • Figure 3 is a representation of E. coli expression, showing that the expression strain can efficiently express the protein of interest regardless of whether it is induced by IPTG.
  • the target protein is present in the precipitate in the form of inclusion bodies;
  • Figure 4 is a purification of the transmembrane peptide-human proinsulin variant recombinant protein, showing that the rinse protein does not contain the protein of interest. As the concentration of imidazole in the eluent increases, the target protein elutes from the nickel column;
  • Figure 5 is a refolding result of the recombinant human proinsulin fusion protein.
  • the mobility of the refolding protein is increased and the position of the band is advanced on the electropherogram compared with the unreconstituted sample. Describe the formation of a disulfide bond, the recombinant protein having a secondary structure;
  • Figure 6 is a schematic diagram showing the secondary structure of a recombinant human proinsulin fusion protein
  • Figure 7 is an immunofluorescence staining detection of recombinant human proinsulin fusion protein through the cell membrane of epidermal cells (HaCaT cells), into the epidermal cells, with the prolongation of incubation time, transmembrane peptide-proinsulin variant recombinant protein in HaCaT cells Constantly gathering;
  • Figure 8 is a C-peptide in which a recombinant human proinsulin fusion protein (molecular weight: 17 kDa) is cleaved by epidermal cells (HaCaT cells) and processed into mature insulin;
  • a recombinant human proinsulin fusion protein molecular weight: 17 kDa
  • Figure 9 is a transdermal absorption of recombinant human proinsulin fusion protein, wherein HA is an Anti-HA tag antibody and DAPI is a nuclear dye. As the incubation time prolonged, the transmembrane peptide-human proinsulin variant recombinant protein gradually passed through the epidermis, and the amount of subcutaneous tissue gradually increased;
  • Figure 10 is a recombinant human proinsulin fusion protein by transdermal administration to lower blood glucose, it can be seen that 30 minutes after administration, the blood glucose of diabetic mice decreased by 60%;
  • Figure 11 is a graph showing that the recombinant human proinsulin fusion protein is hypoglycemic by ocular administration, and it can be seen that the blood glucose of the diabetic mice is decreased by 53% after one hour of administration by eye.
  • the present invention relates to molecular biology experiments, if not specifically noted, may be referred to in "Molecular Cloning” (J. Sambrook, EF Frici, T. Maniartis, Science Press, 1994). . This book and its subsequent published editions are the most commonly used instructive reference books by those skilled in the art in conducting experimental operations related to molecular biology. In addition, according to different experimental purposes, those skilled in the art can carry out corresponding experiments or commissioned specialized companies under the guidance of the operation manual attached to various commercial kits (Kit), such as: gene sequencing, plasmid sequencing and determination. Molecular weight, etc.
  • the reagents used in the present invention were purchased from Sigma-Aldrich if not explicitly indicated.
  • Lentiviral plasmid pCMV-nFM-hppI4-HD (see 201310138167.X) carrying the proinsulin variant encoding gene is used as a template to amplify the proinsulin variant encoding gene by polymerase chain reaction (PCR) according to the expression plasmid pTAT -HA (Addgene) polyclonal cleavage site designed primers, upstream and downstream primers with NcoI and EcoRI cleavage sites.
  • PCR polymerase chain reaction
  • the TA gene was ligated (using a TA-ligation kit, purchased from Quantum Gold Co., Ltd.), and the gene of interest was ligated to the amplified plasmid T vector to amplify the gene plasmid of interest.
  • the expression plasmid pTAT-HA and the plasmid of the target gene were separately extracted, and the two plasmids were digested with restriction endonucleases NcoI and EcoRI. After agarose gel electrophoresis, the target fragment and the vector fragment were separately recovered.
  • the target gene fragment and the expression plasmid vector fragment were ligated with T4 ligase, and transformed into DH5 ⁇ competent state for amplification.
  • the expression plasmid was extracted, and NcoI and EcoRI were digested and identified.
  • the expression plasmid containing the gene encoding the modified proinsulin variant was successfully constructed, and the recombinant plasmid was as shown in FIG. 2 .
  • the expression plasmid of Figure 2 was transformed into an expression host of E. coli (e.g., BL21 (DE3) or BL21 (DE3) plys or Rossetta (DE3), etc.).
  • E. coli e.g., BL21 (DE3) or BL21 (DE3) plys or Rossetta (DE3), etc.
  • the empty vector was transformed into an expression host as a control.
  • control strain and the expression strain were cultured overnight, and collected the next day.
  • the bacterial cell pellet was directly added to the SDS loading buffer and boiled and denatured as a total protein sample.
  • the cells were precipitated, lysozyme lysing bacteria were added, and the supernatant and the precipitate were collected by centrifugation, and SDS loading buffer was separately added.
  • the above extracted protein was identified by 15% SDS-PAGE. As shown in Fig. 3, it can be seen that the expression strain can express the target protein efficiently regardless of whether or not IPTG is induced.
  • the protein of interest is present in the precipitate in the form of inclusion bodies.
  • Example 3 Recombinant human proinsulin fusion protein granules of Escherichia coli expression host fermentation culture
  • Escherichia coli recombinant human proinsulin fusion protein expression vector was cultured overnight in LB medium.
  • the overnight culture was added to the fresh LB medium on the next day, and the culture was expanded at a ratio of 1:1.5 to 100, and the fermentation was continued for 1 hour to 24 hours.
  • the E. coli culture was disrupted by sonication, and the recombinant protein was dissolved in a sterilizing buffer containing a high concentration of a denaturant (8 mol/L urea or 6 mol/L guanidine hydrochloride).
  • the nickel-ion metal chelate agarose was used as the affinity chromatography medium to purify the recombinant protein, and the gradient concentration of imidazole was eluted under denaturing conditions. As shown in Fig. 4, it can be seen that the flow-through liquid does not contain the target protein. The increase in the concentration of imidazole elutes the target protein from the nickel column.
  • FIG. 2 The transmembrane peptide-human proinsulin variant in the denatured state was diluted in a reproducibility buffer by a pulse method at a ratio of 1:100.
  • the non-reduced Tricine-SDS-PAGE showed that the renatured sample slightly moved forward on the electropherogram compared to the denatured sample, indicating that the disulfide bond was formed and the protein had a secondary structure.
  • Figure 6 is a schematic diagram showing the secondary structure of a recombinant human proinsulin fusion protein.
  • Example 6 Recombinant human proinsulin fusion protein penetrates the cell membrane of epidermal cells.
  • 60 wells were inoculated with 60,000 HaCaT epidermal cells per well, 0.5 ml DMEM high glucose medium, and cultured overnight.
  • the cells were blocked for 1 hour at room temperature.
  • the blocking solution is: 1% BSA + 0.1% Triton X-100,
  • Anti-HA tag antibody (1:500 dilution) was added and incubated overnight at 4 °C.
  • the cells were washed with PBS 5 min each time, 3 times.
  • transmembrane peptide-human proinsulin variant recombinant protein gradually increased in HaCaT cells as the incubation time prolonged.
  • Example 7 Epithelial Cells Process recombinant human proinsulin fusion proteins into mature insulin.
  • Anti-HA tag (1:10000) antibody was incubated overnight at 4 °C.
  • Horseradish peroxidase-labeled secondary antibody (1:2000 dilution) was incubated for 1 hour at room temperature.
  • mice of 1C57BL6/J were intraperitoneally injected with 10% chloral hydrate, 300 mg/kg.
  • mice were sacrificed by dislocation, the back skin was removed, rinsed with PBS, and fixed in 10% neutral formalin.
  • Example 9 Recombinant human proinsulin fusion protein is hypoglycemic by transdermal administration.
  • Diabetic model mice were prepared by injecting C57BL6/J mice into 1STZ. Three diabetic mice were taken and intraperitoneally injected with 10% chloral hydrate, 300 mg/kg.
  • Example 10 Recombinant human proinsulin fusion protein is hypoglycemic by ocular administration.
  • Diabetic model mice (C57BL6/J) were injected intraperitoneally with 10% chloral hydrate and 300 mg/kg.
  • the mouse was given an appropriate amount of the fusion protein sample (concentration: 1.744 mg/ml, about 10 ⁇ l) to one eye. After 60 minutes, blood was collected from the tail vein to measure blood glucose levels in diabetic mice. As shown in Figure 11, after 60 minutes of administration of the recombinant human proinsulin fusion protein, the blood glucose of the diabetic mice decreased by 53%.
  • Example 11 Recombinant human proinsulin fusion protein hypoglycemic by sublingual administration.
  • Newborn white rabbits were injected into 1STZ to prepare diabetic model rabbits.
  • Five diabetic rabbits were injected intraperitoneally with 10% chloral hydrate, 600 mg/kg.

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Abstract

La présente invention concerne une protéine de fusion de proinsuline recombinante humaine, comprenant TAT et une proinsuline humaine ayant un site de reconnaissance d'enzyme furine, avec l'acide aminé en position 10 de la chaîne B d'insuline substitué par de l'acide aspartique. La protéine de fusion de proinsuline recombinante humaine fournie par la présente invention peut passer à travers de multiples barrières de membrane biologique et peut atteindre une bioactivité de réduction du glucose sanguin par absorption percutanée, par administration sublinguale et par absorption mucosale.
PCT/CN2018/079375 2017-03-24 2018-03-16 Protéine de fusion de proinsuline recombinante humaine, son procédé de préparation et son utilisation WO2018171535A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
US10799564B1 (en) 2019-05-06 2020-10-13 Baxter International Inc. Insulin premix formulation and product, methods of preparing same, and methods of using same
WO2023057389A1 (fr) 2021-10-04 2023-04-13 Forx Therapeutics Ag Composés inhibiteurs de parg

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* Cited by examiner, † Cited by third party
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CN106749682A (zh) * 2017-03-24 2017-05-31 吉林大学 重组人胰岛素原融合蛋白及其制备方法和用途

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CN1557954A (zh) * 2004-02-10 2004-12-29 北京日出东方科技发展有限责任公司 转导肽-人胰岛素原融合蛋白的表达与应用
CN104211816A (zh) * 2014-09-12 2014-12-17 西安医学院 一种tat-pap融合蛋白及其制备方法和应用
CN106749682A (zh) * 2017-03-24 2017-05-31 吉林大学 重组人胰岛素原融合蛋白及其制备方法和用途

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CN104211816A (zh) * 2014-09-12 2014-12-17 西安医学院 一种tat-pap融合蛋白及其制备方法和应用
CN106749682A (zh) * 2017-03-24 2017-05-31 吉林大学 重组人胰岛素原融合蛋白及其制备方法和用途

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10799564B1 (en) 2019-05-06 2020-10-13 Baxter International Inc. Insulin premix formulation and product, methods of preparing same, and methods of using same
US11033608B2 (en) 2019-05-06 2021-06-15 Baxter International, Inc. Insulin premix formulation and product, methods of preparing same, and methods of using same
US11707509B2 (en) 2019-05-06 2023-07-25 Baxter International, Inc. Insulin premix formulation and product, methods of preparing same, and methods of using same
WO2023057389A1 (fr) 2021-10-04 2023-04-13 Forx Therapeutics Ag Composés inhibiteurs de parg

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