Introduction
1,1,4,7,10,10-Hexamethyltriethylenetetramine may be used as reagent in the synthesis of ideal linear random copolymers containing both vinyl polymer and polyester units in a single polymer chain.1 1,1,4,7,10,10-Hexamethyltriethylenetetramine complexed with CuBr constitutes catalytic complex, used in the copolymerization of poly[ε-caprolactone] with N,N-dimethylamino-2-ethyl methacrylate monomers by atom-transfer radical polymerization (ATRP).2 It may be used as catalyst in the aqueous surface-initiated-ATRP to grow poly(N,N-dimethylacrylamide) (PDMA).3
This Thermo Scientific Chemicals brand product was originally part of the Alfa Aesar product portfolio. Some documentation and label information may refer to the legacy brand. The original Alfa Aesar product / item code or SKU reference has not changed as a part of the brand transition to Thermo Scientific Chemicals.
- Applications
1,1,4,7,10,10-Hexamethyltriethylenetetramine may be used as reagent in the synthesis of ideal linear random copolymers containing both vinyl polymer and polyester units in a single polymer chain.1 1,1,4,7,10,10-Hexamethyltriethylenetetramine complexed with CuBr constitutes catalytic complex, used in the copolymerization of poly[ε-caprolactone] with N,N-dimethylamino-2-ethyl methacrylate monomers by atom-transfer radical polymerization (ATRP).2 It may be used as catalyst in the aqueous surface-initiated-ATRP to grow poly(N,N-dimethylacrylamide) (PDMA).3
2. Acute Health Effects
1. Swallowed!
The material can produce severe chemical burns within the oral cavity and gastrointestinal tract following ingestion. ! Ingestion of alkaline corrosives may produce burns around the mouth, ulcerations and swellings of the mucous membranes, profuse saliva production, with an inability to speak or swallow. Both the esophagus and stomach may experience burning pain; vomiting and diarrhea may follow. ! Accidental ingestion of the material may be damaging to the health of the individual. ! Ingestion of amine epoxy-curing agents (hardeners) may cause severe abdominal pain, nausea, vomiting or diarrhea. The vomitus may contain blood and mucous.
2. Eye!
If applied to the eyes, this material causes severe eye damage. ! Direct eye contact with corrosive bases can cause pain and burns. There may be swelling, epithelium destruction, clouding of the cornea and inflammation of the iris. ! Vapors of volatile amines irritate the eyes, causing excessive secretion of tears, inflammation of the conjunctiva and slight swelling of the cornea, resulting in “halos” around lights. This effect is temporary, lasting only for a few hours. ! The material can produce severe chemical burns to the eye following direct contact. Vapors or mists may be extremely irritating.
3. Skin!
The material can produce severe chemical burns following direct contactwith the skin. ! Skin contact is not thought to have harmful health effects, however the material may still produce health damage following entry through wounds, lesions or abrasions. ! Skin contact with alkaline corrosives may produce severe pain and burns; brownish stains may develop. The corroded area may be soft, gelatinous and necrotic; tissue destruction may be deep. ! Open cuts, abraded or irritated skin should not be exposed to this material. ! Entry into the blood-stream, through, for example, cuts, abrasions or lesions, may produce systemic injury with harmful effects. Examine the skin prior to the use of the material and ensure that any external damage is suitably protected. ! Amine epoxy-curing agents (hardeners) may produce primary skin irritation and sensitization dermatitis in predisposed individuals. Cutaneous reactions include erythema, intolerable itching and severe facial swelling.
4. Inhaled!
The material can cause respiratory irritation in some persons. The body’s response to such irritation can cause further lung damage. ! Inhaling corrosive bases may irritate the respiratory tract. Symptoms include cough, choking, pain and damage to the mucous membrane. ! Inhalation of aerosols (mists, fumes), generated by the material during the course of normal handling, may be damaging to the health of the individual. ! Inhalation hazard is increased at higher temperatures.
5. Chronic Health Effects!
Repeated or prolonged exposure to corrosives may result in the erosion of teeth, inflammatory and ulcerative changes in the mouth and necrosis (rarely) of the jaw. Bronchial irritation, with cough, and frequent attacks of bronchial pneumonia may ensue. Long-term exposure to respiratory irritants may result in disease of the airways involving difficult breathing and related systemic problems. Skin contact with the material is more likely to cause a sensitization reaction in some persons compared to the general population.
Limited evidence suggests that repeated or long-term occupational exposure may produce cumulative health effects involving organs or biochemical systems. There is some evidence that inhaling this product is more likely to cause a sensitization reaction in some persons compared to the general population. Inhalation of epoxy resin amine hardeners (including polyamines and amine adducts) and may produce bronchospasm and coughing episodes lasting several days after cessation of the exposure. Even faint traces of these vapors may trigger an intense reaction in individuals showing “amine asthma”. Both secondary amines and tertiary amines can be nitrosated by nitrosating agents to yield nitrosamines.
In the case of tertiary amines, the mechanism involves intermediate conversion of the tertiary amine to a secondary amine. Some types of nitrosamines have been shown to be carcinogenic in tests using laboratory animals. In general, all nitrosamines are presumed to be carcinogenic unless they’ve been tested and shown to be relatively harmless. Nitrosamines are often generated as undesired byproducts in certain foods (especially bacon) and cosmetic and hygiene products (such as shampoo and hair conditioner).
The occurrence of significant levels of nitrosamines in commercial products is normally not accepted. In formulations which contain nitrosating agents, formation of nitrosamines can become a concern where secondary and/or tertiary amines are used as pH control agents. The most typical nitrosating agents added to formulations are nitrite salts such as sodium or potassium nitrite. Such nitrites are generally added as corrosion inhibitors. Formation of nitrosamine contaminants in such formulations usually results from the reaction of such nitrosating agents with secondary and/or tertiary amines.
Nitrosation can occur during product preparation, either during heating or at moderate temperatures. It can also occur while a product sits on a shelf, especially if the product sits for months in a warehouse that becomes warm. Many efforts have been made to eliminate nitrosamines from various substances, or to reduce their concentrations to the lowest practical levels. In various types of food, the use of ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) and their salts is known to inhibit nitrosamine formation.
3. Method for preparing interferon-polymer conjugate ifn-poegma
1. Abstract
Provided is a method for preparing an interferon-POEGMA conjugate, comprising the following step: polymerizing an interferon-initiator conjugate, OEGMA, CuCl, CuCl2, and 1,1,4,7,10,10-hexamethyltriethylenetetramine in a buffer solution to obtain an IFN-POEGMA conjugate.
2. Technical field
The invention belongs to the field of biomedicine, and particularly relates to a method for preparing an interferon polymer complex IFN-POEGMA.
3. Background technique
Protein has been widely used in many fields such as biomedical development, targeted therapy, and clinical diagnosis. The use of proteins alone has problems such as short half-life and poor stability. The protein-polymer combination is prepared by linking the protein with the polymer, which can effectively improve the solubility, stability, pharmacokinetics and therapeutic efficacy of the protein and reduce its immunogenicity. The traditional method for synthesizing protein-polymer conjugates is to connect pre-prepared polymers with proteins, often with uncertain coupling sites, low efficiency, poor yield, difficult product separation, poor quality control, and difficult activity. Keep a lot of problems.
Interferon-α2 (IFN-α2) is a potent inhibitor of viral replication and tumor cell growth and has been successfully used to treat diseases such as viral hepatitis and cancer. However, the circulatory half-life of IFN after systemic administration is very short, requiring frequent administration and high concentration to achieve the desired therapeutic effect, resulting in some side effects and a heavy economic burden on the patient. Modification of IFN with polyethylene glycol (PEG) is an effective measure to improve its pharmacokinetics and improve its efficacy. It is called long-acting interferon.
However, the current pegylated interferon has disadvantages such as low reaction yield, difficulty in controlling the binding site and coupling stoichiometry, and severely reduced biological activity. For example, Pellet, developed by Schering-Plough, has as many as 14 products, and its activity is only 28%. Pylosin, developed by Roche, has 6 products with an activity of only 7%. The yield of the two products is only About 1%. Therefore, the development of a site-specific modification method with mild reaction conditions, simple steps and high efficiency is particularly important for interferon and other medicinal proteins.
4. Invention disclosure
It is an object of the present invention to provide a process for preparing a protein-polymer combination.
The method provided by the invention comprises the following steps:
The protein-initiator combination, polymer monomer, CuCl, CuCl 2 , 1,1,4,7,10,10-hexamethyltriethylenetetramine are polymerized in a buffer to obtain a protein-polymer. Polymer combination;
The protein-initiator conjugate is a product obtained by covalently linking an initiator to a protein, also known as a macroinitiator.
The above buffer is a PBS aqueous solution having a pH of 7.4 and a concentration of 10 mM, and the specific formulation is: 2.684 g of Na 2 HPO 4 · 12H 2 O, 0.34 g of NaH 2 PO 4 · 2H 2 O, and 8.19 g of NaCl are dissolved in 1 L of water. The solution.
In the above method, the reaction system comprises:
The molar ratio of the protein-initiator combination, the polymer monomer, the CuCl, the CuCl 2 and the 1,1,4,7,10,10-hexamethyltriethylenetetramine is 1:200-10000:10-500:10-2000:10-4000;
a molar ratio of the protein-initiator combination, the polymer monomer, the CuCl, the CuCl 2 and the 1,1,4,7,10,10-hexamethyltriethylenetetramine Is 1:1000-4000:25:75:125;
The coupling ratio of the protein to the initiator in the protein-initiator conjugate is 1:1.
In an embodiment of the present invention, the protein-initiator conjugate is an initiator at the C-terminus of the protein, and the two form a conjugate by forming an amide bond, specifically according to the method comprising the steps of: IFN-LPETGGH6 protein, Sortase AH 6 protein, ATRP initiator 2-bromo-2-methylpropionic acid 2-(2-(2-(2-aminoacetamido)acetamido)acetamido)ethyl ester hydrochloride (AEBM) CaCl 2 is mixed in a pH 7.4 concentration of 50 mM Tris·HCl aqueous solution, and reacted to obtain an interferon-initiator conjugate IFN-Br;
The molar ratio of the above IFN-LPETGGH6 protein, Sortase AH 6 protein, AEBM, and CaCl 2 was 2:1:50:200.
In the above method, the polymerization reaction is carried out under a low oxygen or inert gas atmosphere;
The polymerization time is from 5 minutes to 24 hours, and the temperature of the polymerization is from 0 to 80 °C.
In the above method, in the protein-initiator conjugate, the initiator is attached to the C-terminus of the protein.
In the above method, the protein is an interferon selected from the group consisting of interferon alpha or a fusion protein thereof, interferon beta or a fusion protein thereof, interferon gamma or a fusion protein thereof, interferon lambda or a fusion protein thereof.
In the above method, the amino acid sequence of the interferon alpha fusion protein is sequence 2 in the sequence listing.
In the above method, the initiator is an oligo-glycine functionalized atom transfer radical polymerization (ATRP) initiator;
The oligomeric glycine functionalized atom transfer radical polymerization (ATRP) initiator is specifically 2-(2-(2-(3,4-dibromomaleimide-N-ethoxy)ethoxy) Ethyl)ethyl 2-bromo-2-methylpropionate.
In the above method, the polymer monomer is a water-soluble or biodegradable polymer monomer; the water-soluble or biodegradable polymer monomer is selected from the group consisting of lactic acid, epichlorohydrin, acrylic acid, and methacrylic acid. At least one of styrene, methacrylamide, methyl, acrylamide, norbornene, and oxanorbornene; the polymer is formed by polymerization of the monomer; the polymer is specifically polymethyl Oligoethylene glycol acrylate (OEGMA); the polymer is specifically POEGMA.
The protein-polymer combination prepared by the above method is also within the scope of protection of the present invention.
The use of the above-described protein-polymer combination in the preparation of an antitumor product is also within the scope of the present invention
5. The best way to implement the invention
The experimental methods used in the following examples are conventional methods unless otherwise specified.
The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The plasmid pET-25b(+) in the following examples was a product of Bio-Bioengineering (Shanghai) Co., Ltd.
The TB medium in the following examples was configured as follows: 12 g of peptone, 24 g of yeast extract, and 4 mL of glycerin were added to 900 mL of water, fully dissolved and autoclaved at 121 ° C for 15 min, and the mixture after sterilization was cooled to 60 ° C. Then 100 mL of a sterilized aqueous solution containing 170 mmol/L of KH 2 PO 4 and 0.72 mol/L of K 2 HPO 4 was added.
Human Burkitt’s B lymphoma cells and human ovarian cancer cells (OVCAR-3) in the following examples were purchased from the Chinese Academy of Sciences tumor cell bank.
- The RMPI-1640 medium in the following examples is a Gibco product.
The female athymic (Nude) nude mice in the following examples are products of Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Female athymic (Nude) nude mice are referred to hereinafter as nude mice.
- The flow in the following examples is shown in FIG.
In the following examples, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), microplate reader, dynamic light scattering (DLS), circular dichroism (CD) and other analytical methods were used to characterize the molecular weight of IFN-ELP and IFN. Physical and chemical properties such as phase transition temperature, hydration radius and secondary structure.
Human Burkitt’s B lymphoma cells were used to test the in vitro biological activity of IFN-POEGMA and IFN, ie their ability to resist tumor cell proliferation in vitro; to test the pharmacokinetics of IFN-POEGMA and IFN in vivo using a nude mouse model, using DAS 3.0 Pharmacokinetic analysis software calculates pharmacokinetic parameters; established a nude mouse tumor model to test the anti-tumor effect and drug distribution of IFN-POEGMA and IFN.
The quantitative experiments in the following examples were set to three repetitions unless otherwise specified, and the results were averaged.
8. Claims
- A method for preparing a protein-polymer combination comprises the following steps:
The protein-initiator combination, polymer monomer, CuCl, CuCl 2 , 1,1,4,7,10,10-hexamethyltriethylenetetramine are polymerized in a buffer to obtain a protein-polymer. Polymer combination;
The protein-initiator conjugate is the product obtained by covalent attachment of an initiator to a protein.
- The method according to claim 1, wherein said protein is an interferon, said interferon being selected from the group consisting of interferon alpha or a fusion protein thereof, interferon beta or a fusion protein thereof, interferon gamma or a fusion protein thereof, Interferon lambda or its fusion protein;
Or the protein is an interferon alpha fusion protein as shown in SEQ ID NO: 2 in the SequenceListing.
- The method according to claim 1, wherein said initiator is an initiator for atom transfer radical polymerization, an initiator for reversible addition to a fragmentation chain transfer polymerization, an initiator for ring-opening heterotopic polymerization or an open ring. An addition polymerization initiator; the atom transfer radical polymerization initiator is specifically 2-(2-(2-(2-aminoacetamido)acetamido)acetamide of 2-bromo-2-methylpropanoate Ethyl ester hydrochloride;
The initiator is covalently linked to the C-terminus of the protein.
- The method according to claim 2, wherein the initiator is an initiator for atom transfer radical polymerization, an initiator for reversible addition-break chain transfer polymerization, an initiator for ring-opening heterotopic polymerization or an open ring An addition polymerization initiator; the atom transfer radical polymerization initiator is specifically 2-(2-(2-(2-aminoacetamido)acetamido)acetamide of 2-bromo-2-methylpropanoate Ethyl ester hydrochloride;
The initiator is covalently linked to the C-terminus of the protein.
- The method of claim 1 wherein said polymer monomer is a water soluble or biodegradable polymer monomer; said water soluble or biodegradable polymer monomer is selected from the group consisting of lactic acid, At least one of chlorohydrin, acrylic acid, methacrylic acid, acrylamide, methyl, acrylamide, norbornene, and oxanorbornene; the polymer is formed by polymerization of the monomer The polymer is specifically poly(ethylene glycol oligoethylene glycol).
- The method according to claim 2, wherein said polymer monomer is a water-soluble or biodegradable polymer monomer; said water-soluble or biodegradable polymer monomer is selected from the group consisting of lactic acid, At least one of chlorohydrin, acrylic acid, methacrylic acid, acrylamide, methyl, acrylamide, norbornene, and oxanorbornene; the polymer is formed by polymerization of the monomer The polymer is specifically poly(ethylene glycol oligoethylene glycol).
- The method according to claim 3, wherein said polymer monomer is a water-soluble or biodegradable polymer monomer; said water-soluble or biodegradable polymer monomer is selected from the group consisting of lactic acid, At least one of chlorohydrin, acrylic acid, methacrylic acid, acrylamide, methyl, acrylamide, norbornene, and oxanorbornene; the polymer is formed by polymerization of the monomer The polymer is specifically poly(ethylene glycol oligoethylene glycol).
- The method according to claim 4, wherein said polymer monomer is a water-soluble or biodegradable polymer monomer; said water-soluble or biodegradable polymer monomer is selected from the group consisting of lactic acid, At least one of chlorohydrin, acrylic acid, methacrylic acid, acrylamide, methyl, acrylamide, norbornene, and oxanorbornene; the polymer is formed by polymerization of the monomer The polymer is specifically poly(ethylene glycol oligoethylene glycol).
- The method of claim 1 wherein:
The molar ratio of the protein-initiator combination, the polymer monomer, the CuCl, the CuCl 2 and the 1,1,4,7,10,10-hexamethyltriethylenetetramine is 1:200-10000:10-500:10-2000:10-4000;
Or the molar ratio of the protein-initiator combination, the polymer monomer, the CuCl, the CuCl 2 and the 1,1,4,7,10,10-hexamethyltriethylenetetramine Is 1:1000-4000:25:75:125;
The coupling ratio of the protein to the initiator in the protein-initiator conjugate is 1:1.
- The method of claim 8 wherein:
The molar ratio of the protein-initiator combination, the polymer monomer, the CuCl, the CuCl 2 and the 1,1,4,7,10,10-hexamethyltriethylenetetramine is 1:200-10000:10-500:10-2000:10-4000;
Or the molar ratio of the protein-initiator combination, the polymer monomer, the CuCl, the CuCl 2 and the 1,1,4,7,10,10-hexamethyltriethylenetetramine Is 1:1000-4000:25:75:125;
The coupling ratio of the protein to the initiator in the protein-initiator conjugate is 1:1.
- Method according to claim 1 or 10, characterized in that it comprises:
The polymerization is carried out under a low oxygen or inert gas atmosphere;
The polymerization time is from 5 minutes to 24 hours, and the temperature of the polymerization is from 0 to 80 °C.
- A protein-polymer combination prepared by the method of any of claims 1-10.
- Use of the protein-polymer combination according to claim 12 for the preparation of an antitumor product.
- An antitumor product comprising the protein-polymer combination according to claim 12.