TW202103687A - Powdery composition of ogg1 and uses thereof - Google Patents

Abstract

This invention discloses a powdery composition of OGG1 and a single protecting agent, wherein the single protecting agent is mannitol.

Description

Powdered composition of OGG1 and its use

The present invention relates to a powdered composition comprising OGG1 and a single protective agent, wherein the single protective agent is mannitol.

8-oxoguanine (8-oxoG) lesion [8-oxoguanine (8-oxoG) lesion] is a common DNA lesion. When human individuals are exposed to reactive oxygen species (ROS), ionizing radiation (ionizing radiation) and ultraviolet (ultraviolet, UV) for a long time, they are prone to produce mutagenic base byproducts 8-oxoG , Which in turn can lead to a variety of different diseases, including skin damage, cancer [such as colorectal cancer and lung cancer] and neurological diseases [such as Parkinson's disease) and Alzheimer's disease (Alzheimer's disease)].

8-oxoguanine DNA glycosylase (OGG1) is a DNA repair enzyme (DNA repair enzyme), which is active in both genomic DNA and mitochondrial DNA. Excision repair pathway (base excision repair pathway) and excision of 8-oxoG from damaged DNA, according to which to achieve the effect of treatment and/or prevention of 8-oxoG damage. Therefore, OGG1 has been widely used in cosmetics and medicines.

However, due to its poor stability, OGG1 tends to gradually lose its activity over time. Therefore, relevant researchers in the field have used freeze-drying treatment (lyophilization treatment) to prepare OGG1 into a solid preparation with higher stability, and tried to use appropriate protecting agents to reduce OGG1 activity. Loss. For example, an OGG1 fusion protein (OGG1 fusion protein) produced by Proteintech Group Inc. with the product number Ag7320 is obtained by adding mannitol and trehalose to OGG1 for freeze-drying. OGG1 as a lyophilized powder can be stored at -20°C to -80°C for 12 months.

After research, the applicant unexpectedly found that using mannitol alone as a protective agent to prepare the powdery composition of OGG1 can effectively improve the stability of OGG1, even at room temperature. The storage lasts for up to 24 months.

Summary of the invention

Therefore, in the first aspect, the present invention provides a powdered composition comprising OGG1 and a single protective agent, wherein the single protective agent is mannitol

In the second aspect, the present invention provides a use of the powdered composition as described above for preparing a medicine for treating and/or preventing 8-oxoG injury.

In a third aspect, the present invention provides a method for treating and/or preventing 8-oxoG injury, which comprises administering to an individual in need of treatment and/or prevention of 8-oxoG injury—as described above Powdered composition.

In the fourth aspect, the present invention provides a use of the powdered composition as described above as a cosmetic.

Detailed description of the invention

It should be understood that if any previous case publication is quoted here, the previous case publication does not constitute a recognition: in Taiwan or any other country, the previous case publication forms a common general in the art. Part of knowledge.

For the purpose of this specification, it will be clearly understood that the word "comprising" means "including but not limited to", and the word "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used in this article have meanings commonly understood by those familiar with the art of the present invention. A person familiar with the art will recognize that many methods and materials similar or equivalent to those described herein can be used to implement the present invention. Of course, the present invention is by no means restricted by the described methods and materials.

As used herein, the terms "protein", "polypeptide" and "peptide" can be used interchangeably and mean a polymer composed of amino acid residues, one of which One or more amino acid residues are naturally occurring amino acids or artificial chemical mimics.

Looking for a protecting agent that can make OGG1 more stable, the applicant found through experiments that when lyophilization treatment is used, mannitol is used alone as a protecting agent. The powdered composition of OGG1 can effectively improve the stability of OGG1, thereby extending its shelf life.

Therefore, the present invention provides a powdered composition comprising OGG1 and a single protective agent, wherein the single protective agent is mannitol.

According to the present invention, the powdered composition is obtained by mixing OGG1 with mannitol and then subjecting the resulting mixture to a freeze-drying process to obtain a white powdery dry product.

According to the present invention, the freeze-drying treatment can be carried out using a technique well known and commonly used by those skilled in the art.

It can be understood that the operating conditions of the freeze-drying process will be further changed with factors such as the ratio of OGG1 to mannitol in order to achieve the best extraction effect. The choice of these operating conditions is routinely determined by those who are familiar with the art.

According to the present invention, OGG1 and mannitol are in a ratio ranging from 1:33 (w/w) to 1:2450 (w/w). In a preferred embodiment of the present invention, OGG1 and mannitol are in a ratio of 1:240 (w/w).

According to the present invention, OGG1 can be derived from microorganisms [such as Micrococcus luteus ], plants [such as Arabidopsis thaliana ] or animals [such as Homo sapiens and plankton] OGG1. In a preferred embodiment of the present invention, OGG1 comes from an intelligent person, and is also called human OGG1 (human OGG1, hOGG1).

According to the present invention, hOGG1 can be an alternatively spliced isoforms selected from the group consisting of: hOGG1-1a, hOGG1-1b, hOGG1-1c, hOGG1-2a, hOGG1- 2b, hOGG1-2c, hOGG1-2d, and hOGG1-2e, and combinations thereof. In a preferred embodiment of the present invention, hOGG1 is hOGG1-1a.

According to the present invention, OGG1 can be prepared using genetic engineering techniques well-known and commonly used by those skilled in the art. For example, using recombinant DNA technology (recombinant DNA technology), a nucleic acid coding sequence encoding OGG1 is inserted into an expression vector, and then transformed or transfected into a suitable host In the cell, the nucleic acid coding sequence is then expressed in the host cell, thereby obtaining recombinant OGG1. These techniques can be implemented by those who are familiar with the technology according to their professional qualities, and can refer to the textbooks known in this technology, such as: Molecular Cloning: A Laboratory Manual.

According to the present invention, the purification of the recombinant OGG1 can be performed using a standard protein separation and purification technique well known to those skilled in the art. These standard protein separation and purification techniques include, but are not limited to: salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, Centrifugal filtration, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric point electrophoresis, reverse phase chromatography phase chromatography, anion exchange chromatography, affinity chromatography, and chromatofocusing.

In a preferred embodiment of the present invention, OGG1 is expressed by the rhOGG1 gene (recombinant human OGG1 gene) selected from human ogg1 cDNA clones (product number SC309025, brand OriGen) in E. coli. Purified and obtained.

In addition, OGG1 can also be isolated and purified from a source of microorganisms, plants or animals as described above by using the separation and purification methods commonly used in the art.

Alternatively, OGG1 can also be prepared using traditional chemical synthesis methods well known to chemists, such as using a commercially available synthesis kit or solid phase peptide synthesis.

In addition, the powdered composition is expected to be applicable to the preparation of a medicine for treating and/or preventing 8-oxoG injury.

As used herein, the term "8-oxoG injury" means that any part of a human individual has undergone excessive exposure to reactive oxygen species (ROS), ionizing radiation, and ultraviolet (UV). (overexposure) DNA damage with 8-oxoG mutation caused later, the diseases that it causes include, but are not limited to: skin damage, cancer (such as colorectal cancer and lung cancer). )] and neurological diseases [such as Parkinson's disease, and Alzheimer's disease].

As used herein, the term "treating" or "treatment" 8-oxoG injury means that the severity of the injury or the symptoms of the injury are reduced, or It is the damage that is partially or completely eliminated (eliminated).

As used herein, the term "preventing" or "prevention" 8-oxoG injury means that an individual eliminates or reduces the injury when the injury has not been diagnosed. Incidence, and slow, delay, control or reduce the likelihood or probability of the injury.

According to the present invention, the medicine can be manufactured into a dosage form suitable for topical administration by using techniques well known to those skilled in the art.

According to the present invention, the drug may further include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, decomposers ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, the medicine can be manufactured into an external preparation suitable for topical application to the skin by using techniques well known to those skilled in the art. This includes, but is not limited to: emulsion, Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, Serum, paste, foam, drop, suspension, salve, bandage and the like.

According to the present invention, the external preparation is prepared by mixing the medicine of the present invention with a base well known to those skilled in the art.

According to the present invention, the substrate may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and white Vaseline (white petrolatum), wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ^® 941 (carbopol ^® 941), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)], the activity of Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occluding agents occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants ( propellants) and so on. The selection and quantity of these additives fall within the scope of professionalism and routine technology of those who are familiar with this technology.

The present invention also provides a method for treating and/or preventing 8-oxoG injury, which comprises administering a powdered composition as described above to an individual in need of treatment and/or prevention of 8-oxoG injury.

As used herein, the terms "administering" and "administering" and "application" can be used interchangeably.

According to the present invention, the dosage and frequency of administration of the powdered composition will vary depending on the following factors: the severity of the disease to be improved, the route of administration, and the weight, age, physical condition and response of the individual to be improved. The choice of dosage and frequency of administration falls within the scope of professionalism and routine techniques of those who are familiar with this technology.

In particular, the powdered composition is also expected to be applicable as cosmetics.

According to the present invention, the cosmetic may further include a cosmetically acceptable adjuvant which is widely used in cosmetic manufacturing technology. For example, the cosmetically acceptable adjuvant may contain one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, Chelating agents, surface active agents, coloring agents, thickening agents, fillers, fragrances and odor absorbers. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, the cosmetics can be manufactured into a form suitable for skincare or makeup by using techniques well known to those skilled in the art, which include, but are not limited to: aqueous solution, water -Alcohol solution (aqueous-alcohol solution) or oily solution (oily solution), oil-in-water type (oil-in-water type), water-in-oil type (water-in-oil type) or complex emulsion, gel Glue, ointment, cream, mask, patch, pack, liniment, powder, aerosol, spray, lotion, emulsion, paste, foam, dispersion, drops, mousse ( mousse, sunblock, tonic water, foundation, eyeshadow, makeup remover products, soap, and other body cleansing products.

According to the present invention, the cosmetics can also be used in combination with one or more external use agents with known activity selected from the following: whitening agents (such as tretinoin, catechins) (catechin), koji acid, arbutin and vitamin C], moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts [ Such as aloe extract, skin nutrients, anesthetics, anti-acne agents, antipruritic, analgesic, and anti-dermatitis agents (antidermatitis agents), antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, Antiseborrheic agents, wound-healing agents, corticosteroids and hormones. The selection and quantity of these topical agents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

Detailed description of the preferred embodiment

The present invention will be further described with respect to the following embodiments, but it should be understood that these embodiments are for illustrative purposes only, and should not be construed as limitations on the implementation of the present invention. Examples General experimental materials: 1. The primers used in the following examples for polymerase chain reaction (PCR) were commissioned by Genomics Biosci & Tech. Come on behalf of synthesis. 2. The restriction enzymes used in the following examples were purchased from Promega corporation. 3. The plastid pQE70 used in the following examples was purchased from Qiagen (Valencia, CA). 4. The competent E. coli DH5α cells and the competent E. coli BL21 (DE3) cells [competent E. coli BL21 (DE3) cell] used in the following examples were purchased from the development of probiotics. Company Limited (Yeastern Biotech Co., Ltd.). 5. The mannitol and trehalose used in the following examples were purchased from PanReac and Carbosynth, respectively. 6. The DNA fragments used for OGG1 activity assay in the following examples were synthesized by Sigma-Aldrich. General experimental methods: 1. Unless otherwise specified, the experimental methods used in the present invention [including DNA cloning, PCR, and transformation] are detailed by those skilled in the art Known technology or according to the operating instructions provided by the manufacturer. Example 1. Preparation of the present invention contains a recombinant human-oxo-8- oxoguanine DNA glycosylase (recombinant human 8-oxoguanine DNA glycosylase , rhOGG1) the powdered composition (powdery composition) Experimental Materials: 1. Preparation comprising rhOGG1 Genetically modified E. coli BL21 (DE3) strain:

The selection of rhOGG1 gene is generally carried out according to the method described in Molecular Cloning: A Laboratory Manual. In short, human ogg1 cDNA clones (human ogg1 cDNA clone) (product number SC309025, brand OriGen) were used as a template and one set was used according to NCBI accession number NM_002542.4 [ Homo sapiens for 8 -The nucleotide sequence shown in the transcript variant 1a (transcript variant 1a) of the oxyguanine DNA glycosidase (OGG1) (OGG1) is designed to have a primer pair with the nucleotide sequence shown below (The bottom line indicates the restriction enzyme cutting address) to perform PCR, thereby amplifying a PCR product with rhOGG1 gene. Forward primer rhOGG1-F 5'-aaaa gcatgc gtatgcctgcccgcgcgcttc-3' (Sequence ID: 1) Sph I Reverse primer rhOGG1-R 5'-aaa agatct gccttccggccctttggaac-3' (Sequence ID: 2) Bgl II

青黴素(ampicillin)來進行篩選，繼而抽取出該重組型載體pQE70-rhOGG1，並且利用限制酶切割與定序分析來進行確認。Next, the restriction enzymes Sph I/ Bgl II were used to clone the PCR product into plastid pQE70, and then the resulting recombinant vector pQE70-rhOGG1 was transformed into competent E. coli DH5α cells and used amine

Penicillin (ampicillin) was used for screening, and then the recombinant vector pQE70-rhOGG1 was extracted, and confirmed by restriction enzyme cleavage and sequencing analysis.

青黴素來進行篩選，藉此而得到含有rhOGG1基因的大腸桿菌BL21 (DE3)轉形株。 實驗方法： After that, the confirmed recombinant vector pQE70-rhOGG1 was transformed into competent E. coli BL21 (DE3) cells and used amine

Penicillin was used for screening to obtain a transformed strain of Escherichia coli BL21 (DE3) containing the rhOGG1 gene. experimental method:

First, isopropyl-β-D-thiogalactopyranoside (IPTG) was used to induce the E. coli BL21 ( DE3) The transformed strain expresses rhOGG1 protein, and then a high-pressure cell crusher (model T5/40/EE/GA, brand of Constant systems) is used to break the cells and centrifuge and filter them.

Next, the obtained supernatant was divided into 3 groups, including 1 mannitol group, 1 trehalose group, and 1 mixed group. The supernatant of each group was purified by column chromatography using an AKTA Explorer 100 column chromatograph to sequentially purify the rhOGG1 protein and replace the buffer. The various operating conditions related to column chromatography are shown in Table 1 below. Table 1. Operating conditions of column chromatography purpose Operating parameters condition Purified rhOGG1 Pipe string His XK 50/20 column Detection wavelength UV-280 nm Eluent Buffer A (contains Na ₂ HPO ₄ ‧12H ₂ O, NaCl and H ₃ PO ₄ )/Buffer B [contains Na ₂ HPO ₄ ‧12H ₂ O, NaCl, H ₃ PO ₄ and imidazole] , 70: 30 (v/v) Flow rate (mL/min) 40 Replacement buffer Pipe string Desalting XK 26/G25 string Detection wavelength UV-280 nm Eluent Phosphate buffer Flow rate (mL/min) 13.8

During the elution, when the elution peak reached 15 mAU or more, the eluate was collected in a serum bottle pre-added with an appropriate amount of a protective agent (protecting agent) as shown in Table 2 below, so that each group The eluate contains the same content of protective agent. Table 2. Types and contents of protective agents used in each group Group Protective agent Content (mg/mL) Mannitol Group Mannitol 50 Trehalose group Trehalose 50 Mixed group Trehalose 25 Mannitol 25

After that, a freeze-drying machine (model coolsafe 55-4, brand ScanVac) was used for freeze-drying for 48 hours to obtain a powdery composition containing rhOGG1 as a lyophilized powder. Among them, OGG1 and mannitol are in a ratio of about 1:240 (w/w). Example 2. combination of mannitol and trehalose and mannitol alone as a protective agent for stability rhOGG1 Effect of the powdered composition (Stability) of

In this example, in order to explore the effectiveness comparison of the combined use of different protective agents and the use of a single protective agent in improving the stability of rhOGG1, the applicant will compare the mixed group and mannitol group obtained according to the above example 1 The powdered composition was used for stability test. experimental method:

The powdered composition of each group was subjected to accelerated testing for stability in accordance with the "Drug Stability Test Standards" published by the Food and Drug Administration. Observe the appearance of each group of powdered compositions at the end of the first month after the accelerated stability test and at the end of the sixth month; and before the accelerated stability test (that is, the 0th month) and At the end of the second, fourth, and fifth months after the start of the accelerated stability test, 50 mg of the powdered composition of each group was taken, and then dissolved in 1 mL of pure water as the sample to be tested. It was used for the following OGG1 activity assay (OGG1 activity assay).

The analysis of OGG1 activity is generally carried out with reference to the method described in TW 201531569 A. First, a DNA fragment (hereinafter referred to as DNA fragment 1) having a nucleotide sequence with 8-oxoguanine (8-oxoguanine, 8-oxoG) as shown below (its 5'end and 3' The ends are respectively attached with a fluorescent reporter group (that is, FAM dye) and a fluorescent quencher group (that is, BHQ1)] and one with the following The complementary sequence of DNA fragments (hereinafter referred to as DNA fragment 2) are annealed [the operating conditions are: the reaction at 90°C for 5 minutes, the reaction at 60°C for 10 minutes, and the reaction at 25°C for 10 minutes ], thereby obtaining a DNA substrate. DNA fragment 1 5’-catcgttgtcncagacctggtggat-3’ (Sequence Identification Number: 3) DNA fragment 2 5’-cggtatccaccaggtctgcgacaacgatgaagcc-3’ (Sequence Identification Number: 4) The symbol n stands for 8-oxoG.

Next, add the DNA substrate, 10X reaction buffer (containing 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl ₂ and 10 mM DTT) and S1 nuclease (S1 nuclease) to each group of samples to be tested. ) And mixed, followed by a real time quantitative PCR analyzer (Biometra) at 37°C for 1 hour, and then at 95°C for 1 minute to terminate the enzyme reaction. After the obtained reactant is warmed to 25°C, mix with 99% formamide (containing 0.1% bromophenol blue), and use 20% denaturation containing 7 M urea (urea) Gel (denaturing gel) for electrophoresis.

Then, a luminescence fluorescent imaging system (model 170-8070, brand Bio-Rad) was used to measure the fluorescence intensity of each group of DNA fragments with a size of 25 mer on the gel. And use the analysis software of the system for analysis. In addition, a DNA fragment 1 containing no 8-oxoG was used instead of the DNA substrate to perform the same experiment as a control.

The relative activity of rhOGG1 is obtained by normalizing the fluorescence intensity measured in each group with the fluorescence intensity measured in the control group. result:

By observing the appearance, it was found that the powdered composition of the mixed group had brown lumps due to the browning reaction in the first month after the accelerated stability test was started. In contrast, the mannitol group The powdered composition was still white powder in the 6th month after the accelerated stability test was started.

Figure 1 shows the measured relative activity of rhOGG1 for each group of powdered compositions. It can be seen from Figure 1 that compared with the mannitol group, the relative activity of rhOGG1 in the mixed group will significantly decrease over time. In particular, at the 5th month after the start of the accelerated stability test, the mixed group The relative activity of rhOGG1 in the group has dropped below 40%, while the relative activity of rhOGG1 in the mannitol group is still more than 70%.

This experimental result shows that the powdered composition prepared by using mannitol alone as the protective agent can exhibit an excellent effect in improving the stability of rhOGG1. Example 3. The effect of using mannitol and trehalose alone as protective agents on the stability of rhOGG1 powdered composition

In this example, in order to explore the effectiveness comparison of different types of protective agents in enhancing the stability of rhOGG1, the applicant will use the powdered composition of the mannitol group and the trehalose group obtained according to Example 1 above. Stability test. Experimental method: A. Accelerated test of stability:

The powdered composition of each group is subjected to accelerated stability tests in accordance with the "Drug Stability Test Standards" promulgated by the Food and Drug Administration. At the end of the first month after the start of the accelerated stability test, observe the appearance of each group of powdered compositions; and before the start of the accelerated stability test (that is, the 0th month) and at the start of the stability test At the end of the first, second, and fourth months after the accelerated test, 50 mg of the powdered composition of each group was taken, and then dissolved in 1 mL of pure water as the sample to be tested and carried out Analysis of OGG1 activity as described in Example 2. B. Long-term testing of stability (long-term testing) :

The powdered composition of each group was tested for stability in accordance with the "Drug Stability Test Standards" published by the Food and Drug Administration. Before the start of the long-term stability test (that is, the 0th month) and at the end of the first to 24 months after the start of the long-term stability test, 50 mg of the powdered composition of each group was taken, and then They were dissolved in 1 mL of pure water as samples to be tested and used for the OGG1 activity analysis described in Example 2 above. Results: A. Accelerated test of stability:

By observing the appearance, it was found that the powdered composition of the trehalose group was brown powder due to the browning reaction in the first month after the accelerated stability test was started. In contrast, the powdered composition of the mannitol group was powdered. The composition is still in the form of a white powder.

Figure 2 shows the measured relative activities of rhOGG1 for each group of powdered compositions. It can be seen from Figure 2 that the relative activity of rhOGG1 in the mannitol group was maintained at more than 90% during the 4-month stability accelerated test. In contrast, the relative activity of rhOGG1 in the trehalose group decreased significantly over time. In the fourth month after the accelerated stability test started, it dropped to 50%. B. Long-term stability test:

The results of this experiment show that the relative activity of rhOGG1 in the mannitol group can be maintained at more than 90% during the long-term stability test that lasted 24 months. In contrast, the relative activity of rhOGG1 in the trehalose group is starting to undergo a long-term stability test. In the 7th month thereafter, it dropped to 80% (data not shown).

Based on the above experimental results, the applicant believes that the powdered composition prepared by using mannitol alone as a protective agent can effectively improve the stability of rhOGG1, delay the occurrence of browning reaction, and achieve the effect of extending its shelf life.

All patents and documents cited in this specification are incorporated into this case as reference materials in their entirety. If there is a conflict, the detailed description of the case (including the definition) will prevail.

Although the present invention has been described with reference to the above specific specific examples, it is obvious that many modifications and changes can be made without departing from the scope and spirit of the present invention. Therefore, it is intended that the present invention is only limited by the scope of the patent application attached hereto.

The above and other objectives, features and advantages of the present invention will become apparent with reference to the following detailed description and preferred embodiments and the accompanying drawings, in which: Figure 1 and Figure 2 respectively show the measured relative activities of rhOGG1 for each group of powdered compositions.

Claims (7)

A powdered composition comprising OGG1 and a single protective agent, wherein the single protective agent is mannitol. Such as the powdered composition of claim 1, wherein OGG1 and mannitol are in a ratio ranging from 1:33 (w/w) to 1:2450 (w/w). Such as the powdered composition of claim 2, wherein OGG1 and mannitol are in a ratio of 1:240 (w/w). A use of the powdered composition as claimed in any one of claims 1 to 3 for the preparation of a medicine for treating and/or preventing 8-oxoG injury. The use according to claim 4, wherein the medicine is in a form for topical administration. A use of the powdered composition according to any one of claims 1 to 3 as a cosmetic. The use according to claim 6, wherein the cosmetic is in a form for topical application.

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