CN114470165A

CN114470165A - Application of angiogenin and variant thereof, pharmaceutical composition and preparation of pharmaceutical composition

Info

Publication number: CN114470165A
Application number: CN202210226731.2A
Authority: CN
Inventors: 吴云霞; 蒋宇翔
Original assignee: Wuhan Anjijie Biotechnology Co ltd
Current assignee: Wuhan Anjijie Biotechnology Co ltd
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2022-05-13
Anticipated expiration: 2042-03-09
Also published as: CN114470165B

Abstract

The invention discloses an application of angiogenin and a variant thereof in treating neurodegenerative diseases, such as Parkinson, and belongs to the technical field of biological medicines. The angiogenin can be strongly expressed in the choroid plexus in which ependymal cells and neural stem cells are located, and the angiogenin is also expressed in dopaminergic neurons of the substantia nigra and co-stained with tyrosine hydroxylase in dopaminergic neurons. Meanwhile, PD modeling is carried out on the mouse by using 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine, and then the fact that the PD model mouse is treated by ANG can remarkably relieve PD related symptoms, which is particularly shown in the aspects of recovering the number of nigral dopamine neurons and improving the movement capacity of the mouse.

Description

Application of angiogenin and variant thereof, pharmaceutical composition and preparation of pharmaceutical composition

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of angiogenin and a variant thereof in preparation of a medicine for preventing and/or treating neurodegenerative diseases, a medicine composition and a preparation thereof.

Background

Parkinson's Disease (PD), also known as paralysis agitans, is a common chronic, progressive movement disorder disease of the central nervous system, and belongs to extravertebral system diseases. The clinical symptoms are mainly motional symptoms such as resting tremor, bradykinesia, muscular rigidity, dyskinesia and the like with or without hypomnesis, depression, abnormal smell and the like, which are described by British doctors in James Parkinson 1817 first. The etiology of PD is not clear at present, but it is thought that it is related to genetics, environment, oxidative stress, mitochondrial dysfunction, excitatory amino acid toxicity, etc., and there are many factors that may participate in the pathogenesis.

The main pathological features of PD are loss of nigral dopaminergic neurons, deposition of intracellular lewy bodies of residual neurons, and degeneration of nerve endings in the striatum. With regard to the pathophysiological mechanisms of PD, the theory of dysfunction of nigrostriatal Dopamine (DA) and cholinergic (ACh) neurons is now well recognized. That is, DA energy neurons in the substantia nigra send ascending fiber bundles to new striatum (caudate and putamen), and the terminal synapse with caudate-putamen neurons takes DA as neurotransmitter, and the synapse is directed to the striatum

The GABA (GABA) neurons play an inhibitory role, synapses formed by DA neurons in the caudate nucleus and caudate-putamen neurons take ACh as neurotransmitter to play an excitatory role on GABAergic neurons in striatum, under normal conditions, DA and ACh are in a balance state mutually in opposition and jointly participate in regulating the motor function of the organism, and a PD patient has reduced inhibitory impulsion of the GABAergic neurons in the striatum due to degeneration and damage of the DA ergic neurons in bilateral substantia nigra, while the excitatory impulsion of the ACh on the GABAergic neurons in the striatum is dominant, so that the motor function of the extrapyramidal system is disordered. PD symptoms occur when DA ergic neurons fall to 20% to 40% of normal levels. The anti-PD drugs used in clinic at present are mostly developed according to the theory, namely DA-like drugs or anti-ACh drugs or the combination of the DA-like drugs and the anti-ACh drugs. Other part of the medicine can improve oxidative stress, mitochondrial function and excitatory amino acid toxicity.

In recent years, with the development of technology, attempts to develop molecules that prevent the progress of PD development have been reported, such as bovine knee polypeptides, nucleotide molecules, PD-1 antibodies, and the like.

Angiogenin (ANG), a member of the pancreatic ribonuclease (RNase) superfamily, was originally identified as an angiogenic protein, ANG is widely expressed in humans and has a variety of biological functions, including regulating cell growth and survival, regulating innate immune response, promoting hematopoietic regeneration, promoting neurogenesis, and preventing neurodegeneration. RNA lytic Activity of ANG 10^-5-10^-6Compared to RNase a, however, this low and unique RNase activity is essential for its biological activities in angiogenesis, hematopoietic regeneration and neuronal protection.

Disclosure of Invention

In order to solve the above problems in the prior art, the present invention aims to provide an application of angiogenin and its variant in preparing a medicament for preventing and/or treating neurodegenerative diseases, a pharmaceutical composition and a preparation thereof.

The technical scheme adopted by the invention is as follows: application of angiogenin and its variant in preparing medicine for preventing and/or treating neurodegenerative diseases is provided.

Preferably, the neurodegenerative disease comprises parkinson's disease.

The angiogenin variant can be any sequence with no less than 80% homology to the amino acid change at the following positions of the sequence of human ANG, such as the nucleotide sequence of ANG nuclear localization sequence M30K, M30R, G34K, G34R, M30K G34R, M30R G34K, M30K G34K, etc., or K33A, D116H, Q117G, or the nucleotide sequence of ANG and variants can be used as a gene therapy vector.

A pharmaceutical composition for preventing and/or treating neurodegenerative diseases, comprising the angiogenin and its variant of

claim

1 or 2.

A pharmaceutical formulation for the prevention and/or treatment of neurodegenerative diseases, comprising angiogenin and variants thereof according to

claim

1 or 2, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

Preferably, the effective dosage of the angiogenin and the variant thereof is 10ug/kg/d to 5 mg/kg/d.

Preferably, the carrier is aliphatic polyester polylactic acid and/or polyglycolic acid.

Preferably, the pharmaceutical preparation for preventing and/or treating neurodegenerative disease is administered by inhalation, subcutaneous, intramuscular, intraperitoneal, intravenous, or intracerebral administration.

Preferably, the pharmaceutical formulation is a spray and/or an injection.

The invention has the beneficial effects that:

the present invention provides the use of angiogenin and variants thereof in the treatment of neurodegenerative diseases, such as Parkinson's (PD).

The angiogenin is strongly expressed in the choroid plexus in which ependymal cells and neural stem cells are located, and is also expressed in dopaminergic neurons of the Substantia Nigra (SN), and co-stained with Tyrosine Hydroxylase (TH) in dopaminergic neurons.

The invention simultaneously uses 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) to model the mouse with PD, and further proves that the PD model mouse can obviously relieve PD related symptoms by using ANG treatment, particularly shows that the quantity of nigral dopamine neurons is recovered, and the mouse movement capacity is improved.

Drawings

FIG. 1 is a schematic diagram of SDS-PAGE protein electrophoresis detection of ANG recombinant protein expression in Experimental example 1;

FIG. 2 is a schematic diagram of the ANG recombinant protein in Experimental example 1 before its purification by high performance liquid chromatography;

FIG. 3 is a schematic diagram showing the high performance liquid chromatography purification of ANG recombinant protein in Experimental example 1;

FIG. 4 is a diagram showing the improvement of MPTP-induced motor function of PD mice by ANG in the rotarod experiment in Experimental example 3;

FIG. 5 is a diagram showing that the climbing experiment in Experimental example 4 can improve the MPTP-induced motor function of PD mice in response to ANG;

FIG. 6 is a graph showing that ANG can prevent MPTP-induced SN neuron death in Experimental example 5;

FIG. 7 is a schematic diagram showing the solid counts of ANG's that can prevent MPTP-induced SN neuron death in Experimental example 5;

FIG. 8 is a graph showing the expression of ANG protein at different sites in dopaminergic neurons of SN by immunohistochemical detection in Experimental example 6;

FIG. 9 is a diagram showing the expression of ANG protein at different sites in dopaminergic neurons of SN by immunohistochemical detection in Experimental example 6;

FIG. 10 is a graph showing that ANG is expressed in dopaminergic neurons of SN in Experimental example 6, and that ANG and TH are co-stained by double IF;

FIG. 11 is a graph showing Immunohistochemical (IHC) staining of human ANG in the cortical region of mouse, in experimental example 7, in which intraperitoneal injection of ANG can cross the blood brain barrier to reach the central nervous system;

FIG. 12 is a graph showing the initial Pharmacokinetic (PK) analysis of ANG at different sites in the mouse CNS after intraperitoneal injection in Experimental example 8;

FIG. 13 is a schematic diagram showing the operation of angiogenin and its variants in the present invention.

Detailed Description

The present invention is further illustrated below with reference to specific examples. It will be appreciated by those skilled in the art that the following examples, which are set forth to illustrate the present invention, are intended to be part of the present invention, but not to be construed as limiting the scope of the present invention. The reagents used are all conventional products which are commercially available.

Example 1:

and (4) preparing recombinant ANG. Coli BL21(DE3) transformed with plasmid pET11 α -rhaNG was selected as a single colony, inoculated into 2mL of LB medium containing 100ug/mL of ampicillin, shaken well, and subjected to amplification overnight, and the overnight culture was further added to 0.5L of 2 XYT (1.6% trypton, 1% yeast extract, 0.5% NaCl, pH7.0) culture medium and further subjected to amplification culture in a 37 ℃ incubator. And (3) detecting a small amount of the bacterial liquid by using an ultraviolet spectrophotometer at regular intervals, adding 0.5mmol of Isopropyl-beta-D-thiogalactoside (IPTG) when A600 is detected to be 0.8-1.0, culturing for 3-5 hours in a constant temperature shaking table at 37 ℃, and allowing escherichia coli for enough time to induce the expression of rANG.

The cells were disrupted by an ultrasonic cell disrupter, centrifuged at 12000rpm for 20 minutes, the supernatant was discarded, and the inclusion body precipitate was collected.

The inclusion body precipitate is dissolved in 20mL of inclusion body denaturation liquid (7mM guanidine hydrochloride, 0.15MGSH, 0.1MHcl,2mM EDTA, pH8.0), stirred and dissolved for 2h in an inert gas nitrogen environment, centrifuged at 12000rpm at 4 ℃ for 20 min, the supernatant is collected to obtain denatured protein liquid, the denatured protein liquid is slowly dripped into 500mL of renaturation liquid (0.6mM GSSG, 0.5M L-argine, pH8.0, filtered by a 0.22um microporous filter before use), the mixture is continuously stirred for 1h after all the components are added, and the mixture is kept standing overnight. The next day the renaturation solution was diluted to 2L and stored at 4 ℃. Packing the column with a gel packing of a cation affinity chromatography column soaked in 20% ethanol, equilibrating the column with buffer A (10mM Tris-HCl, 0.2M NaCl, pH8.0), loading the renatured protein onto the column, after sufficient binding, eluting with buffer B (10mM Tris-HCl, 0.8M NaCl, pH8.0), and then concentrating and collecting the protein with a 10KD ultrafiltration centrifuge tube.

Further, Waters 1525 hplc, C18 reverse hplc column (250mm × 4.6mm) gradient separation purification rhANG, Waters 2487 detector 280nm wavelength monitoring chromatography elution flow.

Composition of mobile phase chromatography solution A: 0.1% TFA, composition of chromatography solution B: a solution of isopropanol, acetone and water containing 0.08% TFA 3:2:2, and a gradient elution method, wherein the flow rate is 0.8mL/min, and the flow rate is 0-20 minutes, and 70% solution A → 55% solution A; 55% solution A → 50% solution A in 20-40 min; 50% of solution A after 40-50 minutes; 50-70 min 50% solution A → 70% solution A, the peak appears at about 29min, the peak protein is collected and freeze-dried at-80 ℃ for later use.

20 μ L of IPTG-induced bacterial liquid, protein liquid passing through a cation affinity chromatographic column and peak protein solution collected by HPLC are respectively taken and put into a centrifugal tube, 5 μ L of 5 xSDS loading buffer is added for denaturation, protein is separated by 15% SDS-PAGE, and Coomassie brilliant blue is used for staining.

As shown in FIG. 1, SDS-PAGE was prepared for rhaNG. In the figure: 1. inducing the bacterial liquid with 1mmol/L IPTG to obtain inclusion body protein; 2. washing the purified protein; 3. purifying the protein by Sepharose column chromatography; 4. and (4) a control group.

The results show that: SDS-PAGE protein electrophoresis detects the expression result data of the ANG recombinant protein, and the expression result data are at 15KD and at 6 KD.

As shown in fig. 2, the results show that: schematic diagram before high performance liquid chromatography purification of ANG recombinant protein,

as shown in fig. 3, the results show that: the high performance liquid chromatography of the ANG recombinant protein is shown in the figure after purification,

example 2: preparing a PD animal model and carrying out administration treatment.

9 week old C57BL6 mouse, 3 days before the dosing, carry out a series of behavioural training to the mouse every day such as commentaries on classics stick, pole climbing, select the mouse that the motion ability is at the same level and be used for the model building, avoid the difference of individual motion, the mouse divide into 3 groups: i.e. control group, MPTP model group, MPTP + ANG treated group, 10 animals per group. MPTP was intraperitoneally injected at an interval of 2 hours at 12mg/kg/d for 3 consecutive molding, and PBS or ANG protein (10. mu.g/mouse) was intraperitoneally injected daily for 7 consecutive days from the next day.

Example 3: rod rotation experiment

The time that the mouse stays on the rotating rod can reflect the movement function of the mouse. Mice were first trained in an accelerated mode (4-40 rpm over 5 minutes) twice daily for 2 consecutive days. Training was then performed in a constant speed mode (16rpm) and the mice were allowed to rest on the rotarod for over 300 s. The stay time of the rotating rod is detected before administration, mice with different time differences are removed, and experimental errors are reduced.

As shown in fig. 4, the results show that: ANG treatment significantly increased the rod-turning time, indicating that ANG could improve MPTP-induced motor function in PD mice.

Example 4: pole climbing experiment

A climbing rod with the diameter of 1cm and the length of 50cm is manufactured, a small wood ball with the diameter of 1.5cm is fixed at the top end of the climbing rod, gauze is wound on a straight rod to increase friction, the vertical rod is straightened, a mouse is placed on the small ball at the top end of the climbing rod, the time of the mouse returning to the bottom of the rod from the beginning to the turning head is recorded, the detection interval is 5 minutes every time, and the average value is obtained after 3 times of detection. 3 days before administration, the mice are trained to climb the rods every day, and the mice with different rod climbing time are removed, so that experimental errors are reduced.

As shown in fig. 5, the results show that: ANG treatment significantly reduced pole climbing time, indicating that ANG can improve MPTP-induced motor function in PD mice.

Example 5: TH expression and positive cell counts in SN at substantia nigra.

TH positive cells were counted stereoscopically throughout the area, and the image shown was representative of each group (the most dense part). Counting was performed by serial sections of the entire SN, every 4 sections per animal. Data are expressed as mean ± SE.

As shown in fig. 6 and 7, the results show that: the number of TH-positive cells in the group without MPTP treatment and with PBS treatment was 5824. + -.68. The number of TH positive cells treated with MPTP in combination with PBS was 992. + -. 104. In the MPTP + ANG treatment group, the number of TH positive cells is 2044 +/-196, and the survival rate of TH positive neurons is doubled by ANG treatment.

Example 6: immunohistochemistry and fluorescence immunohistochemistry detect the expression of ANG proteins at different sites of the central nervous system.

As shown in fig. 8 and 9, the results show that: the ANG protein is strongly and highly expressed in the choroid plexus part and the ventricular membrane part where the neural stem cells are positioned by immunohistochemical detection.

As shown in fig. 10, the results show that: ANG was expressed in dopaminergic neurons of SN, and double IF showed ANG co-staining with TH.

Immunohistochemistry step: and (3) putting the brain tissue slices into an oven at 72 ℃ for half an hour, putting the brain tissue slices into turpentine oil, incubating for 3-5 minutes, sequentially adding 100%, 95% and 75% alcohol to water for 5 minutes each time, and washing the slices twice by dH2O for 5 minutes each time. Immersed in 1 × citrate antigen retrieval solution (98 deg.C) for about 10 minutes, cooled to room temperature, and the sections washed three times with dH2O for 5 minutes each. Incubate in 3% aqueous hydrogen peroxide for 10 minutes and wash the sections twice with dH2O for 5 minutes each. Sections were blocked with 100ul goat serum blocking solution at room temperature for 30 min, the blocking solution was removed, 50ul Tyrosine Hydroxylase (TH) primary antibody diluted 1:200 in PBS was added, and incubated overnight at 4 ℃. Equilibrating at room temperature for 30 minutes, removing the antibody solution, washing the sections with PBS 3 times for 5 minutes each; 50ul of secondary antibody was added dropwise to the sections and incubated at room temperature for 30 minutes. The secondary antibody was washed 3 times with PBS for 5 minutes each; mixing DAB staining solution A and B according to the ratio of 1: mix at 1 ratio, dilute with PBS, mix well, apply 100ul dab to each section and observe closely, stop staining when appropriate staining intensity is achieved. Sections were washed twice with dH2O for 5 minutes each, 100ul hematoxylin was added dropwise to each section, stained for 2 minutes, and sections were washed twice with dH2O for 5 minutes each. The slices were differentiated in alcohol containing 1% hydrochloric acid for about 3 seconds, and the slices were washed twice with dH2O for 5 minutes each. Sections were dehydrated twice in 75%, 95%, 100% alcohol for 5 minutes each time. After the slices are naturally dried, the neutral gum is used for sealing the slices. Fluorescence immunohistochemistry step: performing same fluorescence immunohistochemistry, simultaneously incubating two primary antibodies, namely TH primary antibody (1:200) and ANG monoclonal antibody 26-2F (2ug/ml), wherein the secondary antibody is a fluorescent secondary antibody, the red is TH, the green is ANG, the secondary antibody is incubated for 30 minutes in a dark place, then washing is performed for 3 times, and then 50% glycerol mounting is immediately performed, and a photograph is observed and taken under a Leica fluorescence microscope. The same applies to the immunohistochemical method below.

Example 7: 10 μ g/mouse ANG was intraperitoneally injected, and the animals were sacrificed 2 hours later and isotype-matched non-immune IgG, i.e., a schematic using PBS, was detected using monoclonal antibody (mAb)26-2F from ANG as a negative control. Intraperitoneal injected ANG can cross the blood brain barrier to reach the central nervous system, Immunohistochemical (IHC) staining of human ANG in the mouse cortical areas.

As shown in fig. 11, the results show that: no signal was detected in the same concentration of specimens, indicating immunohistochemical specificity.

Example 8: 11-week-old mice were injected intraperitoneally with ANG (10. mu.g/mouse), sacrificed at different times after cardiac perfusion, and the amount of ANG in different CNS tissues was determined by ELISA. Primary pharmacokinetic analysis of ANG at different sites of the mouse central nervous system after intraperitoneal injection.

As shown in fig. 12, the results show that: ANG was detected in the brainstem, cerebellum, mesencephalon, hippocampus, hypothalamus and spinal cord 0.5 hours after injection and stabilized between 5-6 hours. Human ANG was detectable in the cortex after 3 hours, and also stabilized at 6 hours. After 6 hours of intravenous injection, human ANG concentrations in the nigrostriatal body reached 100pg/mg tissue, 2.5-4 times higher than in other brain tissues.

The ELISA detection method comprises the following steps: the kit comprises the following steps:

1. weighing tissue specimen, adding 0.5mg of the weighed tissue specimen into 1ml of PBS (2-8 ℃), fully homogenizing the tissue specimen by using a hand or a homogenizer, centrifuging for about 20 minutes (3000 r/min at 2000-.

2. Diluting standard substance at 1000ng/L, 500ng/L, 250ng/L, 125ng/L, 62.5 ng/L.

3. Sample adding: and blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way), standard holes and sample holes to be detected are respectively arranged. The standard sample is accurately loaded on the ELISA plate by 50 mul, and the sample to be detected is loaded in the sample hole by 40 mul of sample diluent, and then 10 mul of sample to be detected is loaded (the final dilution of the sample is 5 times). Adding sample to the bottom of the plate hole of the enzyme label, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall.

4. And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.

5. Preparing liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.

6. Washing: carefully uncovering the sealing plate film, discarding liquid, drying, filling washing liquid into each hole, standing for 30 seconds, discarding, and repeating for 5 times.

7. Adding an enzyme: 50 μ l of enzyme-labeled reagent was added to each well, except for blank wells.

8. And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.

9. Washing: carefully uncovering the sealing plate film, discarding liquid, drying, filling washing liquid into each hole, standing for 30 seconds, discarding, and repeating for 5 times.

10. Color development: adding 50 μ l of color-developing agent A and 50 μ l of color-developing agent B into each hole, shaking gently and mixing, and developing for 10 minutes at 37 ℃ in a dark place.

11. And (4) terminating: the reaction was stopped by adding 50. mu.l of stop solution to each well.

12. And (3) determination: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank well being zeroed. The measurement should be performed within 15 minutes after the addition of the stop solution.

13. And (3) calculating: drawing a standard curve on coordinate paper by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate, and finding out the corresponding concentration from the standard curve according to the OD value of the sample; multiplying by dilution times; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the OD value, substituting the OD value of the sample into the equation to calculate the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the concentration of the sample.

The principle of operation of angiogenin and its variants is shown in figure 13:

the survival and anti-apoptotic functions of ANG are via hydrolysis to produce a class of biologically active small RNAs, tRNA-derived stress-induced small RNAs (tirnas), which inhibit overall protein translation but allow Internal Ribosome Entry Sequence (IRES) -mediated translation, an anti-apoptotic and survival-promoting gene-preferred protein translation mechanism. When cells are stressed, for example under oxidation, hypoxia and stress, ANG is transferred to stress particles in the cytoplasm, which contain untranslated mRNA and RNA binding proteins that form a stop protein translation response upon cellular stress, thereby conserving anabolic energy and promoting cell survival under adverse conditions.

The present invention is not limited to the above alternative embodiments, and any other products in various forms can be obtained by the present invention, and the present invention is within the protection scope of the present invention. The above embodiments should not be construed as limiting the scope of the present invention, and it will be understood by those skilled in the art that modifications may be made to the technical solutions described in the above embodiments, or equivalent substitutions may be made to some or all of the technical features thereof, without departing from the scope of the present invention, and at the same time, such modifications or substitutions may not make the essence of the corresponding technical solutions depart from the scope of the embodiments of the present invention.

Claims

1. An application of angiogenin and its variant in preparing the medicines for preventing and/or treating the neurodegenerative diseases is disclosed.

2. Use of angiogenin and variants thereof according to claim 1 in the manufacture of a medicament for the prevention and/or treatment of neurodegenerative disorders including parkinson's disease.

3. A pharmaceutical composition for preventing and/or treating neurodegenerative diseases, comprising the angiogenin and its variant of claim 1 or 2.

4. A pharmaceutical formulation for the prevention and/or treatment of neurodegenerative diseases, comprising angiogenin and variants thereof according to claim 1 or 2, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

5. The pharmaceutical preparation for preventing and/or treating neurodegenerative disease according to claim 4, wherein the angiogenin and its variant are used in an effective dose of 10ug/kg/d to 5 mg/kg/d.

6. The pharmaceutical preparation for the prevention and/or treatment of neurodegenerative disease according to claim 5, wherein the carrier is aliphatic polyester polylactic acid and/or polyglycolic acid.

7. The pharmaceutical preparation for preventing and/or treating neurodegenerative disease according to claim 6, wherein the pharmaceutical preparation for preventing and/or treating neurodegenerative disease is administered by inhalation, subcutaneously, intramuscularly, intraperitoneally, intravenously or intracerebrally.

8. The pharmaceutical preparation for preventing and/or treating neurodegenerative disease according to claim 7, wherein the pharmaceutical preparation is a spray and/or an injection.