CN111110857A - Long-acting sustained-release polyethylene glycol modified antitumor drug and preparation method thereof - Google Patents
Long-acting sustained-release polyethylene glycol modified antitumor drug and preparation method thereof Download PDFInfo
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- 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
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- A—HUMAN NECESSITIES
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- 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
- A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6907—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a long-acting slow-release polyethylene glycol modified anti-tumor drug and a preparation method thereof, wherein the long-acting slow-release polyethylene glycol modified anti-tumor drug comprises catechol structure functionalized polyethylene glycol used for site-specific modification of anti-tumor protein, and the preparation method comprises the following steps: s1, preparing catechol structure functionalized polyethylene glycol; s2, modifying the antitumor protein by using the catechol structure functionalized polyethylene glycol in a fixed-point manner; and S3, purifying and separating to improve the slow release effect of the PEG protein drug and maintain stable blood concentration for a long time.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a long-acting slow-release polyethylene glycol modified anti-tumor medicine and a preparation method thereof.
Background
With the development of biotechnology, protein biopharmaceuticals are being developed and applied more and more widely due to their advantages of high activity, strong specificity, clear biological function, low toxicity, etc. However, problems such as short half-life, poor stability, poor water solubility, immunogenicity, and a fast renal clearance rate have limited their further development. In order to solve these problems, researchers have made various attempts, and polyethylene glycol modification of protein drugs is one of the most important chemical modification techniques. Polyethylene glycol modification, also known as pegylation, is a process of attaching polyethylene glycol polymers to some biomolecules (such as peptides, proteins and antibody fragments), and aims to improve the problems of the protein drugs, and a series of polyethylene glycol derivatives have been developed for this purpose.
The modification of the PEG to the protein antitumor drug can effectively reduce the immunogenicity of the protein drug, change the half-life of the drug, and the controllable release PEG derivative is essentially characterized in that a degradable chemical bond is introduced between the PEG and a target molecule, so that the modified product can be degraded (such as enzymolysis, hydrolysis and the like) under certain conditions after entering the human body, the protein drug is released, the activity of the protein drug is maintained, the half-life is prolonged, and the sustained release rate is improved.
Most of the PEG protein drugs in the prior art are products which carry out random non-site-specific modification on free amino groups on the surface of protein molecules, for example, CN201310154747.8, "a method for determining modification sites of polyethylene glycol-modified protein drugs", determines the effects of polyethylene glycol on different sites of modified protein, although polyethylene glycol successfully modifies protein drugs and does not change the activity thereof, the half-life prolonging effect is not good, on the one hand, because the chemical bond between the amino group of protein and PEG is weaker, the protein drugs are easy to degrade in a short time; on the other hand, the protein has more amino acid residues on the surface, so that modification sites are too many, modified products are not uniform, the modification rate is not high, part of unmodified protein drugs directly act in a body, the concentration of the drugs with the slow release effect is reduced, and the drugs need to be frequently taken to maintain the blood concentration.
Therefore, the development of a novel PEG fixed-point modified drug protein is necessary to prolong the slow release effect of the PEG fixed-point modified drug protein.
Disclosure of Invention
The invention provides a long-acting sustained-release polyethylene glycol modified antitumor drug and a preparation method thereof, which can improve the sustained-release effect of a PEG protein drug and maintain stable blood concentration for a long time.
In order to solve the technical problems, the invention adopts the technical scheme that the long-acting slow-release polyethylene glycol modified anti-tumor drug comprises catechol structure functionalized polyethylene glycol used for site-specific modification of anti-tumor protein, reactive groups on the surface of the protein are mostly nucleophilic, and the nucleophilic activity is from large to small, namely, sulfydryl is more than α -amino is more than epsilon-amino is more than carboxyl, so that the sulfydryl is the best reaction site of the drug protein, and the modified polyethylene glycol can perform addition reaction with the sulfydryl of the protein by modifying the polyethylene glycol, so that the bond energy is strong, the degradation condition is high, the time is long, the degradation rate is slow, and the slow-release effect is good.
Preferably, the molecular weight of the polyethylene glycol is 5-20 kDa, if the molecular weight of the polyethylene glycol is too large, the modified protein drug has reduced hydrolytic activity because the polyethylene glycol chain length is increased and steric hindrance is increased, and if the molecular weight of the polyethylene glycol is too small, the modified protein drug does not have the function of reducing the immunogenicity of the protein;
preferably, the anti-tumor protein contains cysteine, the existence of cysteine can prevent catechol structure functionalized polyethylene glycol from directly acting on a disulfide bond of the protein, so that the tertiary structure of the protein is influenced, cysteine can be directly used as a target point for a protein medicament containing cysteine to act on the catechol structure functionalized polyethylene glycol, free cysteine can be transformed by a genetic engineering means if the protein medicament does not contain cysteine, and the research of transforming some amino acids which do not influence the activity of the protein into cysteine by site-directed mutagenesis is relatively mature, so that the details are not repeated in the scheme.
Further, a preparation method of the long-acting slow-release polyethylene glycol modified anti-tumor drug is provided, which comprises the following steps:
s1, preparing catechol structure functionalized polyethylene glycol;
s2, modifying the antitumor protein by using the catechol structure functionalized polyethylene glycol in a fixed-point manner;
and S3, purifying and separating.
Preferably, the preparation of the catechol-structure-functionalized polyethylene glycol in S1 includes one of a Schiff base preparation method and a trichlorotriazine linking method, and the catechol-structure-functionalized polyethylene glycol is prepared in multiple ways to prove that the modification effect on the protein is independent of other substances.
Preferably, the modification in S2 has a pH of 7.4, a modification time of 1-3h, and a modification temperature of 25-35 deg.C, and because it is a modification of protein, conditions such as isoelectric point of protein, adaptation temperature of protein, etc. should be considered to prevent inactivation of protein.
Preferably, the purification method of S3 includes one or more of dialysis, ultrafiltration concentration, desalination method, and ion exchange separation method, the pegylated protein is purified by stages, for the separation of polyethylene glycol in the reaction solution after modification, the oligomeric polyethylene glycol is dialyzed, the polymeric polyethylene glycol is separated by ultrafiltration concentration, desalination method, and ion exchange method, the excess polyethylene glycol is removed, and the modification rate of the target molecule is ensured;
preferably, the schiff base preparation method comprises the following steps of taking sodium cyanoborohydride as a reducing agent, and carrying out schiff base reaction on 3, 4-dihydroxybenzaldehyde and methoxy polyethylene glycol amine to obtain the catechol structure functionalized polyethylene glycol, and the specific steps can comprise: dissolving 3, 4-dihydroxy benzaldehyde in hot absolute ethyl alcohol, regulating pH to 5-6, introducing inert gas, stirring and uniformly mixing, weighing methoxy polyethylene glycol amine, dissolving in hot absolute ethyl alcohol, adding into a round-bottom flask in three times, heating the reaction mixture to 80 ℃ for reflux reaction, adding sodium cyanoborohydride, continuously refluxing, performing suction filtration, performing rotary evaporation, adding deionized water for dissolution, and dialyzing.
Preferably, the trichlorotriazine connecting method comprises the following steps of taking trichlorotriazine as a connecting agent, connecting methoxypolyethylene glycol with dopamine, and specifically comprises the steps of mixing trichlorotriazine with raw materials, adding glacial petroleum acid, reacting at 20-40 ℃, performing suction filtration, performing vacuum drying to obtain an intermediate product, mixing dopamine hydrochloride with the intermediate product, stirring for reacting, performing suction filtration, dissolving with water, and performing dialysis to obtain the product.
Preferably, the reaction temperature of the trichlorotriazine linking method is 20-40 ℃, three chlorine atoms of trichloro-triazine crop have different reactivity, under alkaline conditions, the first chlorine can react with a basic group or an amino group at 4 ℃, after the first chlorine reacts, the second chlorine needs room temperature to react, and after the second chlorine reacts, the third chlorine needs at least 80 ℃ to react, so that the grafting reaction can be controlled by controlling the reaction conditions.
The principle of the scheme is that the catechol-structure functionalized polyethylene glycol has a site-specific recognition function and can react with amino groups or sulfhydryl groups of compounds in the presence of weak alkali or oxidizing agents in a Schiff base or addition manner, the polyethylene glycol with the catechol structure can preferentially react with sulfhydryl groups of proteins, so that the polyethylene glycol is specifically modified on the surfaces of the proteins to form single-modified and uniform pegylated proteins, reactive groups on the surfaces of the proteins are mostly nucleophilic, and the nucleophilic activity is from large to small, wherein the sulfhydryl groups are more than α -amino groups more than epsilon-amino groups more than carboxyl groups more than hydroxyl groups, so that the sulfhydryl groups are the best reaction sites of the drug proteins, the bond energy is strongest, the degradation time in vivo is longer, and the PEG concentration is high, the blood concentration can be stably maintained, and the sustained-release effect is good because the PEG is modified at a fixed point, the modification rate is high and uniform, and most of protein drug molecules are modified.
The beneficial effect of this scheme lies in:
1. the synthesized polyethylene glycol with the catechol structure can preferentially react with sulfhydryl groups of proteins, the polyethylene glycol is specifically modified on the surfaces of the proteins to form single-modified and uniform pegylated proteins, the modification rate is high, most protein drug molecules are modified, and therefore the single-modified and uniform pegylated proteins have a slow release function, the drug concentration which has a slow release effect with the pegylated protein in unit mass is high, the blood drug concentration can be stably maintained, and the slow release rate is improved;
2. because the derivatized PEG is combined with the sulfydryl of the protein, the bond energy is strongest, the drug protein is bonded with the catechol, the PEG layer is wrapped layer by layer to form micelles at the periphery, the degradation time in the organism is longer, the slow release rate is high, frequent medicine taking is not needed, and the pain and the economic burden of a patient are relieved;
3. the scheme carries out segmented purification on the PEG protein, for the separation of polyethylene glycol in reaction liquid after modification, the oligomerization polyethylene glycol adopts a dialysis method, the high-polymer polyethylene glycol adopts ultrafiltration concentration, a desalination method and an ion exchange method for separation, redundant polyethylene glycol is removed, and the modification rate of target molecules is ensured;
4. the preparation process is simple, safe and nontoxic;
drawings
FIG. 1 shows the results of the modification rate and half-life of the modified proteins obtained in examples and comparative examples.
Detailed Description
The present invention is described in detail below with reference to the following examples and the accompanying drawings, and it should be noted that the following examples are only for illustrative purposes and should not be construed as limiting the scope of the present invention.
Example 1
Preparing pyrocatechol structure functionalized polyethylene glycol by using Schiff base reaction:
taking a three-neck flask, dissolving 0.1g of 3, 4-dihydroxybenzaldehyde in 20ml of hot anhydrous ethanol (containing anhydrous sodium sulfate), adjusting the pH to 5-6 with dilute hydrochloric acid, introducing inert gas, stirring uniformly, weighing 0.5g of methoxypolyethylene glycol amine (a commercial product, CAS number 147867-65-0) to be dissolved in the hot anhydrous ethanol, adding the solution into the round-bottom flask in three times, heating the reaction mixture to 80 ℃ for reflux reaction, adding sodium cyanoborohydride, continuing to reflux, filtering off the anhydrous sodium sulfate, placing the filtrate into the round-bottom flask, rotatably evaporating the anhydrous ethanol, adding deionized water for dissolution, dialyzing in a dialysis bag (molecular weight cut-off 3500) with deionized water for three days and three nights, and carrying out vacuum freeze drying to obtain a white solid, namely the product.
Example 2
Synthesizing catechol-structure functionalized oligoethylene glycol by using trichlorotriazine as a connecting compound:
the three chlorine atoms of trichlorotriazine have different reactivity, under the alkaline condition, the first chlorine can react with hydroxyl or amino at 4 ℃, after the first chlorine reacts, the second chlorine needs room temperature to react, and after the second chlorine reacts, the third chlorine needs at least 80 ℃ to react, so that the grafting reaction can be controlled by controlling the reaction condition to be 20-40 ℃.
Adding 0.5g of trichlorotriazine into 100ml of anhydrous benzene for full dissolution, adding 5g of raw material methoxy polyethylene glycol (5 kDa) into the solution, uniformly stirring in a round-bottom flask, reacting at room temperature overnight, adding 75ml of iced petroleum acid into the reaction solution, reacting at 20-40 ℃ to generate a large amount of white flocculent precipitate, filtering off the solution by suction, washing the obtained white solid with benzene for three times, drying in vacuum to obtain white powder as an intermediate product, weighing 0.3g of dopamine hydrochloride, dissolving in 50ml of dioxane, introducing inert gas, adding 3g of the intermediate product into 100ml of dioxane for full dissolution, dripping into the solution by using a dropping funnel, stirring and reacting at room temperature for 5 hours, adding 200ml of iced petroleum ether into the reaction solution to generate a large amount of white flocculent precipitate, filtering off the solution, dissolving the obtained white solid with water, and filling into a dialysis bag with the molecular weight cut-off of 3500, dialyzing with water for three days and three nights, and vacuum freezing for drying to obtain the product.
Example 3
Synthesizing the catechol structure functionalized medium polyethylene glycol by using trichlorotriazine as a connecting compound:
adding 0.1g of trichlorotriazine into 50ml of anhydrous benzene for full dissolution, adding 2g of raw material methoxy polyethylene glycol (12 kDa) into the solution, uniformly stirring in a round-bottom flask, reacting overnight at room temperature, adding 50ml of iced petroleum ether into the reaction solution, reacting at 20-40 ℃ to generate a large amount of white flocculent precipitate, filtering off the solution by suction, washing the obtained white solid with benzene for three times, drying in vacuum to obtain white powder as an intermediate product, weighing 0.1g of dopamine hydrochloride, dissolving in 30ml of dioxane, introducing inert gas, adding 1g of the intermediate product into 100ml of dioxane for full dissolution, dripping into the solution by a dropping funnel, stirring and reacting for 5 hours at room temperature, adding 200ml of iced petroleum ether into the reaction solution to generate a large amount of white flocculent precipitate, filtering off the solution, dissolving the obtained white solid with water, and filling into a dialysis bag with the molecular weight cut-off of 3500, dialyzing with water for three days and three nights, and vacuum freezing for drying to obtain the product.
Example 4
Synthesizing pyrocatechol structure functionalized high polyethylene glycol by using trichlorotriazine as a connecting compound:
adding 0.05g of trichlorotriazine into 30ml of anhydrous benzene for full dissolution, adding 2g of raw material methoxy polyethylene glycol (20 kDa) into the solution, uniformly stirring in a round-bottom flask, reacting overnight at room temperature, adding 40ml of iced petroleum ether into the reaction solution, reacting at 20-40 ℃ to generate a large amount of white flocculent precipitate, filtering off the solution by suction, washing the obtained white solid with benzene for three times, drying in vacuum, taking the obtained white powder as an intermediate product, weighing 0.1g of dopamine hydrochloride, dissolving in 30ml of dioxane, introducing inert gas, adding 1.2g of the intermediate product into 100ml of dioxane for full dissolution, dripping into the solution by using a dropping funnel, stirring for reaction for 5 hours at room temperature, adding 100ml of iced petroleum ether into the reaction solution to generate a large amount of white flocculent precipitate, filtering off the solution by suction, dissolving the obtained white solid with water, filling into a dialysis bag with the molecular weight cut-off of 3500, dialyzing with water for three days and three nights, and vacuum freezing for drying to obtain the product.
Example 5
Catechol-structure functionalized oligo-polyethylene glycol modified drug protein and purification:
adding 5mg of catechol-structure-functionalized oligo-ethylene glycol described in example 1 or example 2 into the diluted protein solution, wherein the molar ratio is about 30 times, fully dissolving the polymer by vortex oscillation, adjusting the pH value to 7.4, carrying out shaking table reaction for 1-3h at 25-35 ℃, dialyzing the product for 24h by a 20mMPB (pH 7.4) dialysis bag with molecular weight cut-off of 8000-14000, and obtaining the catechol-structure-functionalized oligo-ethylene glycol modified drug protein.
Example 6
Modifying the drug protein by polyethylene glycol in catechol structure functionalization and purifying:
adding 5mg of the catechol-structure functionalized medium polyethylene glycol described in example 3 into the diluted protein solution, wherein the molar ratio is about 30 times, fully dissolving the polymer by vortex oscillation, adjusting the pH to 7.4, carrying out shaking table reaction for 1-3h at 25-35 ℃, and desalting the product by a 5ml desalting column (SephadexG-25) to obtain the catechol-structure functionalized low polyethylene glycol modified drug protein.
Example 7
Catechol structure functionalized high polyethylene glycol modified drug protein and purification:
adding 5mg of catechol-structure-functionalized high-polyethylene glycol described in example 4 into the diluted protein solution, wherein the molar ratio is about 30 times, fully dissolving the polymer by vortex oscillation, adjusting the pH to 7.4, carrying out shaking table reaction for 1-3h at 25-35 ℃, and desalting the product by a 5ml desalting column (SephadexG-25) to obtain the catechol-structure-functionalized low-polyethylene glycol modified drug protein.
Comparative example 1
Adding 5mg of a commercially available modifier mPEG-succinimide carbonate into the diluted protein solution, wherein the molar ratio is about 30 times, fully dissolving the polymer by vortex oscillation, adjusting the pH to 7.4, carrying out shaking table reaction for 1-3h at the temperature of 25-35 ℃, and dialyzing the product for 24h by a 20mMPB (pH 7.4) dialysis bag with the molecular weight cutoff of 8000-14000 to obtain the non-site-specific amino modified drug protein.
Comparative example 2
Adding 5mg of a commercially available modifier PEG-hydrazide into the diluted protein solution, wherein the molar ratio is about 30 times, performing vortex oscillation to fully dissolve the polymer, adjusting the pH to 7.4, performing shaking table reaction at 25-35 ℃ for 1-3h, and desalting the product by using a 5ml desalting column (SephadexG-25) to obtain the catechol structure functionalized low polyethylene glycol modified drug protein.
Example 7
The modification rates of examples 1 to 6 were measured by photometric method, and the PEG modification rates were calculated by measuring the changes in the numbers of free epsilon-amino groups/thiol groups on the protein surface before and after modification, in this example, the modification rates were calculated by measuring the change numbers of thiol groups by the national standard DTNB method for examples 5 to 7, and the modification rates were calculated by measuring the change numbers of amino groups by the national standard TNBS method for comparative examples 1 to 2, and the results are shown in fig. 1.
Example 8
Animal experiments are carried out on the modified proteins obtained in examples 5-7 and comparative examples 1-2, and the time required for half of the drug concentration in animal plasma is measured, and the result is shown in figure 1, so that the modified protein prepared by the scheme has longer half-life and good slow-release effect.
The contents of the present invention are not limited to the above-described embodiments, and other embodiments within the technical teaching of the present invention by those skilled in the art are within the scope of the present invention.
Claims (10)
1. The long-acting sustained-release polyethylene glycol modified antitumor drug is characterized by comprising catechol structure functionalized polyethylene glycol for site-specific modification of antitumor protein.
2. The long-acting sustained-release polyethylene glycol modified antitumor drug according to claim 1, wherein the molecular weight of the polyethylene glycol is 5kDa-20 kDa.
3. The long-acting sustained-release polyethylene glycol-modified antitumor drug according to claim 1, wherein the antitumor protein contains cysteine.
4. A preparation method of a long-acting slow-release polyethylene glycol modified antitumor drug is characterized by comprising the following steps:
s1, preparing catechol structure functionalized polyethylene glycol;
s2, modifying the antitumor protein by using the catechol structure functionalized polyethylene glycol in a fixed-point manner;
and S3, purifying and separating.
5. The method for preparing a long-acting sustained-release polyethylene glycol modified antitumor drug according to claim 4, wherein the step of preparing the catechol-structure functionalized polyethylene glycol in S1 comprises one of a Schiff base preparation method and a trichlorotriazine linking method.
6. The preparation method of the long-acting sustained-release polyethylene glycol modified antitumor drug according to claim 4, wherein the pH value modified in S2 is 7.4, the modification time is 1-3h, and the modification temperature is 25-35 ℃.
7. The method for preparing a long-acting sustained-release polyethylene glycol modified antitumor drug according to claim 4, wherein the purification method of S3 comprises one or more of dialysis, ultrafiltration concentration, desalination method and ion exchange separation method.
8. The preparation method of the long-acting sustained-release polyethylene glycol modified antitumor drug according to claim 5, characterized in that the Schiff base preparation method comprises the following steps of taking sodium cyanoborohydride as a reducing agent, and carrying out Schiff base reaction on 3, 4-dihydroxybenzaldehyde and methoxy polyethylene glycol amine to obtain the catechol-structure functionalized polyethylene glycol.
9. The preparation method of the long-acting sustained-release polyethylene glycol modified antitumor drug according to claim 5, wherein the trichlorotriazine coupling method comprises the following steps of taking trichlorotriazine as a coupling agent and coupling methoxypolyethylene glycol with dopamine.
10. The method for preparing a long-acting sustained-release polyethylene glycol modified antitumor drug according to claim 9, wherein the reaction temperature of the trichlorotriazine coupling method is 40-60 ℃.
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Application publication date: 20200508 |