Patent Application
20240269160
This application claims priority to and the benefit of Chinese patent Application No. 202310099279.2 filed Feb. 2, 2023. The entire contents of Chinese patent Application No. 202310099279.2 and the English translation of Chinese patent Application No. 202310099279.2 are incorporated herein by reference.
This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing .xml file entitled “000006us_SequenceListing.xml”, file size 3,254 bytes, created on Jan. 17, 2024. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).
The present invention relates to a field of drug, in particular to use of citicoline in regulating respiratory chain function related protein level and preparing drug for preventing and treating respiratory chain dysfunction.
A respiratory chain is a continuous reaction system composed of a series of hydrogen transfer reactions and electron transfer reactions arranged in a certain order. It hands off paired hydrogen atoms from metabolites to oxygen to produce water, while ATP is produced.
NDUFA8, UQCRC2, and COXIV are known respiratory chain function related proteins.
1,2-diacyl-sn-glycero-3-phosphocholine (PC) is an amphipathic molecule composed of a hydrophilic head and a hydrophobic tail. It is a type of phospholipid with a choline group inserted at the head. The 1,2-diacyl-sn-glycero-3-phosphocholine is an important component of biofilms.
Phosphatidylethanolamine (PE), also known as brain phospholipids, is one of the important phospholipids that form biofilms. It mainly exists in the brain, nerves, microorganisms, and soybeans, playing important roles in signal transduction and maintaining life functions.
PC and PE also play important roles in respiratory chain function. Respiratory chain dysfunction is mainly manifested as a decrease in content of PC, PE, and ATP in body at the molecular level. Respiratory chain dysfunction can lead to various diseases.
Therefore, it is necessary to develop new drugs for preventing and treating respiratory chain dysfunction in this field.
Citicoline is a nucleoside derivative. The reported therapeutic uses of Citicoline include improving consciousness state and electroencephalogram of consciousness disorders after head trauma or brain surgery, promoting recovery of upper limb motor function in stroke hemiplegic patients, and having a certain effect on promoting brain function recovery and awakening.
However, there have been no reports in this field on use of citicoline in regulating respiratory chain function related protein level, preventing and treating respiratory chain dysfunction.
Based on above requirements and research gaps in this field, the present invention provides use of citicoline in regulating respiratory chain function related protein level and preparing drug for preventing and treating respiratory chain dysfunction.
The technical solution of the present invention is as follows:
Use of citicoline in regulating respiratory chain function related protein level.
Use of citicoline in preparing drug for preventing and treating respiratory chain dysfunction.
The drug comprises: pharmacodynamically active ingredients; The pharmacodynamically active ingredients include: citicoline;
A shRNA causing respiratory chain dysfunction, its reverse complementary sequence is shown as SEQ ID NO. 2.
DNA sequence corresponding to the shRNA is shown as SEQ ID NO. 1;
Use of shRNA in preparing a respiratory chain dysfunction animal model, a reverse complementary sequence of shRNA is shown as SEQ ID NO. 2.
DNA sequence corresponding to the shRNA is shown as SEQ ID NO. 1;
A method for preparing a respiratory chain dysfunction animal model, comprising the following step: downregulating levels of NDUFA8 and/or UQCRC2 and/or COXIV in animal by shRNA whose reverse complementary sequences is shown as SEQ ID NO. 2.
The respiratory chain dysfunction refers to content of PC and/or PE and/or ATP in animal body decreased;
On the one hand, the present invention provides a use of citicoline in regulating respiratory chain function related protein level.
On the other hand, the present invention provides a use of citicoline in preparing drug for preventing and treating respiratory chain dysfunction.
In some countries or regions where patent laws permit, the present invention also claims use of citicoline for preventing and treating respiratory chain dysfunction.
Optionally, the respiratory chain dysfunction is caused by decreased expression of NDUFA8, UQCRC2, and COXIV.
Optionally, the drug is a drug that improves respiratory chain dysfunction.
Improving metabolism can be reflected in an increase in content of phosphatidylcholine (PC) and phosphatidylethanolamine (PE).
Improving respiratory chain function can be reflected in an increase in ATP content.
Optionally, the drug may be capsule, tablet, granule, pill, or oral liquid.
Optionally, the drug also includes pharmaceutically acceptable excipients or additives.
Above drug can significantly increase the expression of NDUFA8 protein, UQCRC2 protein, and COXIV protein.
The present invention also provides a method for treating and/or preventing and/or alleviating and/or improving respiratory chain dysfunction, including administering citicoline to recipient animals for treatment and/or prevention and/or alleviation and/or improvement of respiratory chain dysfunction.
Optionally, the respiratory chain dysfunction is caused by decreased expression of NDUFA8, UQCRC2, and COXIV.
A respiratory chain dysfunction animal model caused by decreased expression of NDUFA8, UQCRC2, and COXIV mentioned above can be a shRNA intervention mice, whose levels of NDUFA8, UQCRC2, and COXIV proteins significantly decrease. For example, a recombinant adeno-associated virus (rAAV) vector carrying shRNA is introduced into mice through tail vein injection.
The shRNA refers to a hairpin structure nucleic acid that reduces mRNA content or reduces protein levels through RNA interference technology.
Optionally, the shRNA intervention in mice includes using an rAAV vector carrying shRNA to reduce expression of NDUFA8, UQCRC2, and COXIV proteins in the recipient mice.
The present invention proposes for the first time use of citicoline in treating respiratory chain dysfunction, which can improve respiratory chain dysfunction. The respiratory chain dysfunction is caused by downregulation of NDUFA8 and/or UQCRC2 and/or COXIV levels in animal by shRNA with reverse complementary sequences as shown in SEQ ID NO. 2, specifically manifested as a decrease in PC, PE, and ATP levels in the animal body. Through experiments, it was found that after treatment with citicoline, levels of PC, PE, and ATP in respiratory chain dysfunction animal were increased, while the decreased levels of NDUFA8, UQCRC2, and COXIV proteins were restored. It's demonstrated that, citicoline can reverse molecular level symptoms of respiratory chain dysfunction and can be used as a drug for treating respiratory chain dysfunction.
Marks in the figures are listed as follows:
rAAV-TNT shCON: mice injected with the control vector
rAAV-TNT-control; The control vector rAAV-TNT-control is the commercially available empty vector (pAAV-TNT vector).
rAAV-TNT-shRNA: mice injected with rAAV vector rAAV-TNT-shRNA carrying shRNA;
rAAV-TnT-shCON+control: blank control group mice injected with control vector rAAV-TNT-control at 8 weeks of age, and fed with physiological saline at 12 weeks of age;
rAAV-TnT-shRNA+control: positive control group mice injected with rAAV vector rAAV-TNT-shRNA carrying shRNA at 8 weeks of age, and fed with physiological saline at 12 weeks of age;
rAAV-TnT-shCON+CDP-choline: control group mice injected with the control vector rAAV-TNT-control at 8 weeks of age, and then fed with cytosine diphosphate choline at 12 weeks of age;
rAAV-TnT-shRNA+CDP-choline: experimental group mice injected with rAAV vector rAAV-TNT-shRNA carrying shRNA at 8 weeks of age, and then fed with choline diphosphate at 12 weeks of age.
The technical solution and technical effects of the present invention will be described clearly and completely in the following content by combining with specific examples and experimental examples. Obviously, the described experimental examples and examples are only a part of the present invention's experimental examples and examples, not all of them. Based on the experimental examples and examples of the present invention, all other examples obtained by a person skilled in the art without creative work fall within protection scope of the present invention.
The experimental methods in the following embodiments and experimental examples, unless otherwise specified, are conventional methods and are carried out according to the techniques or conditions described in literatures in this field or in accordance with product manuals. The materials, reagents, etc. used in the following experimental examples can be commercially available unless otherwise specified.
Structural formula of citicoline in the following experimental example is formula 1, with CAS number 987-78-0. It is a product of MedChemExpress company (product number HY-B0739).
Data was processed using GraphPad Prism 8 statistical software, and the experimental results were presented as mean±standard error using a two tailed t-test.
Nanodrop® ND-1000 nucleic acid analyzer
ABI 9700 PCR instrument
ABI 7900HT fluorescence real-time quantitative PCR instrument
Beckman X-15R Low Temperature High Speed Centrifuge
TRIZOL was purchased from Invitrogen;
The reverse transcription kit was purchased from Thermofish Company; SYBR Green was purchased from Thermofish Company;
EasyPure Plasmid MiniPrep Kit plasmid extraction kit was purchased from Beijing TransGen Biotech Company;
PAAV-TNT vector, DH5a competent cell and 293T cell (human embryonic renal epithelial cell) used in experiment example 1 can be commercially obtained;
C57BL/6 mice used in experiment example 2 and experiment example 3 were from Jiangsu GemPharmatech biotechnology Company Limited;
This group of examples provides use of citicoline in regulating respiratory chain function related protein level.
In specific examples, said respiratory chain function related protein is selected from groups consisting of NDUFA8, UQCRC2. COXIV.
In some embodiments, said regulating refers to increasing levels of NDUFA8 and/or UQCRC2 and/or COXIV in respiratory chain dysfunction animal model;
A person skilled in the art, teached by the present invention, can use citicoline to regulate levels of respiratory chain function related proteins other than NDUFA8, UQCRC2, and COXIV. Any use of citicoline for upregulating, downregulating, positive regulating, negative regulating, reducing, increasing, enhancing other respiratory chain function related protein level and/or knocking down, knocking out, silencing, or overexpressing genes of other respiratory chain function related protein falls within protection scope of the present invention.
This group of example provides a use of citicoline in preparing drug for preventing and treating respiratory chain dysfunction.
In specific embodiments, the drug comprises: pharmacodynamically active ingredients; the pharmacodynamically active ingredients include: citicoline;
Preferably, the drug further comprises: pharmaceutically acceptable excipients;
In specific embodiments, the pharmaceutically acceptable excipients are selected from: solvents, projectiles, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavor correction agents, preservatives, suspension aids, coating materials, aromatics, anti adhesives, integrators, penetration enhancers, pH regulators, buffering agents, plasticizers, surfactants, foaming agent, defoaming agent, thickener, packaging agent, moisturizing agent, absorbent, diluent, flocculant, anti flocculant, filter aid, release blocker.
In specific embodiments, said preparing is selected from groups consisting of synthetizing, amplifying, expressing, cloning, secreting, enriching, expanding, and reaction generating;
In specific embodiments, the effective concentration for alleviating and/or improving respiratory chain dysfunction with citicoline is 500 mg/kg.
According to production needs and practical requirements, and based on teachings of the present invention, a person skilled in the art can add various pharmaceutically acceptable excipients/excipients to citicoline, to prepare various dosage forms (including but not limited to oral liquid, injection, tablet, powder, granule, aerosol, spray, etc.) for easy sales or promotion.
Any use of citicoline in preparing drug or product for treating respiratory chain dysfunction, and/or any use of citicoline for treating and/or relieving and/or improving of respiratory chain dysfunction, and/or any action of placing of citicoline in packaging boxes labeled with therapeutic/relief/improvement effects for respiratory chain dysfunction, falls within protection scope of the present invention.
Examples Group 3. shRNA of the Present Invention
This group of examples provides a shRNA causing respiratory chain dysfunction. All examples of this group possess the following common features: its reverse complementary sequence is shown as SEQ ID NO. 2.
In specific examples, DNA sequence corresponding to the shRNA is shown as SEQ ID NO. 1;
Above shRNAs are all designed and synthesized for the first time according to the present invention, and any act of amplifying, synthetizing, producing, manufacturing, selling, promising for sale, using, importing, exporting, secreting, expanding, enriching, connecting, transforming, cloning, expressing any of above shRNAs, and/or any act of using any of above shRNAs for pharmaceuticals or as pharmaceutical ingredients, and/or, Any use of any of above shRNAs for treating falls within the protection scope of the present invention.
Examples Group 4. Use of shRNA of the Present Invention
This group of examples provides use of shRNA in preparing a respiratory chain dysfunction animal model. All examples of this group possess the following common features: a reverse complementary sequence of shRNA is shown as SEQ ID NO. 2.
In some examples, DNA sequence corresponding to the shRNA is shown as SEQ ID NO. 1;
Any behavior of using reverse complementary sequences of shRNA such as SEQ ID NO. 2 to prepare respiratory chain dysfunction models, and/or any behavior of using reverse complementary sequences of shRNA such as SEQ ID NO. 2 to cause respiratory chain dysfunction, and/or any behavior of placing reverse complementary sequences of shRNA such as SEQ ID NO. 2 in packaging boxes labeled with respiratory chain dysfunction, falls within the scope of protection of the present invention.
This group of examples provides a method for preparing a respiratory chain dysfunction animal model. All examples of this group possess the following common features: comprising the following step: downregulating levels of NDUFA8 and/or UQCRC2 and/or COXIV in animal by shRNA whose reverse complementary sequences is shown as SEQ ID NO. 2;
Synthesizing shRNA and its reverse complementary sequence separately, and dissolving them with TE.
Nuclease-Free Water 36 μl
Annealing Buffer for DNA Oligos (5×) 10 μl
DNA oligo A (50 μM) 2 μl
DNA oligo B (50 μM) 2 μl
90° C. for 3 minutes, 37° C. for 1 hour, and 4° C. for storage.
The eukaryotic expression vector pAAV-TNT was digested with BamH I and Not I at 37° ° C. for 2 hours with the following reaction system:
10×K Buffer 1 μl
BSA 1 μl
BamHI 1 μl
Not I 1 μl
PAAV-TNT 2 μl
ddH2O 14 μl
Using 1% agarose electrophoresis gel to electrophoresis products of double enzyme digestion, and then using the agarose gel DNA recovery kit of TaKaRa Agarose Gel DNA Purification Kit Ver manufactured by TaKaRa Corporation. 2.0 to recover the products of the double enzyme digestion. The specific operation steps are as follows:
Selecting monoclonal colonies, adding them to 3 ml of LB liquid medium containing Amp+, and shaking at 37ºC for 280 rpm overnight. Extracting the plasmid using the EasyPure Plasmid MiniPrep Kit from Beijing TransGen Biotech Company. The specific steps are as follows:
Identifying the constructed plasmid by double enzyme digestion and sequencing, to obtain the eukaryotic expression plasmid pAAV-TNT-shRNA. Its structure is shown in
Preparing a 1 L sterile conical flask, adding 300 ml sterile LB medium, and adding ampicillin solution to a final concentration of 100 μg/ml. Adding 50 μl of the desired plasmid (pXX9; phelper; pAAV-TNT-shRNA) and incubating overnight at 280 rpm and 37° C. Extracting the plasmid according to instructions of the OMEGA E.Z.N.A.® Endo-Free Plasmid Maxi Kit. The specific steps are as follows:
Growing 293T cells (human embryonic kidney epithelial cells) to 90%, at 1-2 hours before calcium and phosphate transfection, replacing each culture dish with 12-15 ml fresh medium (containing serum). Adding calcium chloride (CaCl2) to a 50 ml centrifuge tube, then adding the plasmid to form a Ca-DNA mixture. Mixing thoroughly, slowly adding 2×HEBS BUFFER to the Ca-DNA mixture to form a Ca-DNA-P mixture, and shaking the centrifuge tube while adding 2×HEBS. Mixing thoroughly to form calcium phosphate particles. After 8-12 hours, replacing with 18-20 ml serum-free medium. After 72 hours, aspirating and discarding the medium, washing 3 times with PBS, adding 1 ml Tris+NaCl (pH 8.5) to each culture dish, scraping the cells with a spatula, collecting them in a clean centrifuge tube, and freezing at −80° C.
Removing cells frozen at −80° C., thawing and dissolving them at 37° C., freeze-thawing repeatedly for 4 times, centrifuging at 8,000 g for 15 min, and placing supernatant into a clean centrifuge tube, discarding the cell pellet.
Mixing thoroughly ethanol precooled to −20° C. with rAAV at a volume ratio of 3:1. After placing in refrigerator at −20° C. for 2 hours, centrifuging at 13,000 rpm for 15 minutes at 4ºC, and discarding the supernatant. After the ethanol evaporates, adding the corresponding volume of Tris+NaCl (pH 8.5) to dissolve the precipitate. Filtering with a Millipore small filter (0.22 μm).
Sample processing: 40 μl of rAAV-TNT-shRNA or rAAV-TNT-control virus solution
Proteinase K (20 mg/ml) 5 μl 55° C., reaction for 1 hr;
Phenol: chloroform: isoamyl alcohol 45 μl
Centrifuge at 4° C. for 5 minutes at 12,000 g to recover aqueous phase;
Chloroform 45 μl
Centrifuge at 4ºC for 5 minutes at 12,000 g to recover aqueous phase.
Real-time PCR:
Primer 1 (10 μm) 0.4 μl
Primer 2 (10 μm) 0.4 μl
SYBR Green I Mix 10 μl
ddH2O 8.2 μl
Template 1 μl
95° C. 30 sec-(95° C. 5 sec-60° C. 5 sec-72° C. 20 sec)×40 cycles-Melting Curve
Using 8-week-old C57 mice, injecting rAAV-TNT-shRNA or rAAV-TNT-control through tail vein, with a viral titer of 1×1011 PFU per mouse. Collecting organ tissues until the end of this experiment (12 weeks later). Placing 5 mg of tissue in a centrifuge tube and adding 500 μL of RIPA lysis buffer (pre-added protease inhibitor and phosphatase inhibitor). Homogenizing tissue thoroughly by using a homogenizer. Placing the homogenized tissue on ice for 10 minutes and placing the centrifuge tube in an ultrasonic disrupter for ultrasonic disruption. Then, centrifuging it at 12000 g/min for 20 minutes at 4° C., and aspirating supernatant into another clean centrifuge tube. Measuring protein concentration by using BCA method. Obtaining a standard curve by using the measured standard concentration to calculate protein concentration of sample. Using Western blot to detect expression of NDUFA8 protein, UQCRC2 protein, and COXIV protein, with GAPDH protein as a control. The results are shown in
Analyzing phospholipid metabolism content by targeted ultra-high performance liquid chromatography-mass spectrometry quantitative analysis: weighing about 20 mg of mouse tissue, adding grinding beads to grind thoroughly, adding 70 μL of internal standard (purchased from Avanti Polar Lipids Alabaster, USA), each of which was 100 ng, and dissolving in chloroform/methanol solution (1:1, volume ratio), and 10 μL of BHT solution dissolved in methanol (concentration of 50 mg/mL) to obtain liquid. Then, adding 1 mL of chloroform/methanol solution (1:1, volume ratio) to the liquid at room temperature for 2 minutes to obtain solution. Then, adding 0.45 mL of ultrapure water to the solution, and using a tissue grinder to homogenize. Centrifuging the homogenized liquid at 4ºC for 5 minutes, and collecting the lower layer liquid into a new EP tube. After completing above steps, extracting the upper residue again with chloroform/methanol solution (1:1, volume ratio) and ultrapure water. Combining the two collected lower layer liquids, drying them, and adding 80 μL of chloroform/methanol solution (1:1, volume ratio) to reconstitute them. After filtering with a 0.22 μM filter membrane, entering the detection. The on-machine detection and analysis were completed by Black Technology Co., Ltd. Name was described by Lipid Map (http://www.lipidmaps.org/). ATP content detection: adding 200 μL of lysis buffer per 20 mg of tissue, and then homogenizing the tissue. After homogenization, centrifuging at 12000 g for 5 min at 4ºC, and retaining the supernatant for use. In advance, adding 100 μL of ATP detection working solution to enzyme-labeled tubes, and after allowing the detection working solution to fully consume ATP with placing for 5 minutes, adding 100 μL the collected tissue lysis supernatant samples and standard samples as ATP detection working solution per tube as soon as possible using a pipette gun. Rapidly mixing, determining RLU value by using a chemiluminescence instrument, and drawing a standard curve based on the RLU value and standard samples. Standardizing ATP concentration of each sample based on protein concentration and simultaneously detecting protein concentration.
It's shown by results that, PC and PE (
C57 mice were randomly divided into four groups (rAAV-TnT-shCON+control agent, rAAV-TnT-shRNA+control agent, rAAV-TnT-shCON+CDP-choline, and rAAV-TnT-shRNA+CDP-choline). At 8 weeks of age, injecting the corresponding rAAV virus through tail vein. At 12 weeks of age, feeding mice with 500 mg/kg CDP-choline and control agent (saline) every other day. At the end of the experiment (12 weeks later), collecting heart tissues, and detection indicators were the same as in experimental example 2.
It's shown by results that, compared with the rAAV-TnT-shCON+control group mice, expression levels of NDUFA8 protein, UQCRC2 protein, and COXIV protein (
| Number | Date | Country | Kind |
|---|---|---|---|
| 202310099279.2 | Feb 2023 | CN | national |
