CN112206314A

CN112206314A - Transferrin and application of transferrin and mannolite sodium combined capsule

Info

Publication number: CN112206314A
Application number: CN202010706434.9A
Authority: CN
Inventors: 栗琳; 谢蓝; 张丽丽; 丁亮
Original assignee: Jiangsu Haosi Muke Biotechnology Co ltd
Current assignee: Jiangsu Haosi Muke Biotechnology Co ltd
Priority date: 2020-04-27
Filing date: 2020-07-21
Publication date: 2021-01-12
Also published as: CN112206315A; CN111514282B; CN111514282A

Abstract

The invention provides an application of transferrin and transferrin combined mannitol sodium capsules. The method specifically comprises the following steps: use of transferrin or transferrin in combination with mannolite sodium capsules for the preparation of a medicament for the prevention or/and amelioration or/and treatment of mild cognitive impairment. The invention has the beneficial effects that: transferrin can improve the cognitive level of patients with mild cognitive dysfunction, and transferrin is used together with the mannate sodium capsules to further improve the effect of the mannate sodium capsules on improving the cognitive level of patients with mild cognitive dysfunction.

Description

Transferrin and application of transferrin and mannolite sodium combined capsule

Technical Field

The invention belongs to the application field of biological medicines, and particularly relates to transferrin, and a combination use of the transferrin and a mannate sodium capsule.

Background

The Mantidine sodium capsule (GV-971, trade name "Jiu phase one") is a low molecular acid oligosaccharide compound extracted from marine brown algae. The medicine is the first new medicine for treating Alzheimer Disease (AD) targeting the brain-intestinal axis in China. Mannitol sodium capsules are approved conditionally for marketing with their excellent safety. Previously, the world-wide treatment of AD has relied primarily on 5 drugs previously marketed, all of which have been primarily directed at delaying the progression of and ameliorating clinical symptoms of early AD. The theory supporting new drug tests against AD suggests that β -amyloid deposits in the brain trigger neuroinflammation, which in turn damages neurons, leading to AD. However, clinical test results of the drug developed by targeting beta-amyloid show that the clinical efficacy of the drug is not obvious, and the reason of the drug is probably related to single targeting of a new drug and complex pathogenesis of AD.

Donepezil is a second-generation cholinesterase (ChE) inhibitor, which can obviously inhibit ChE in brain tissues and is clinically used for treating mild and moderate dementia, but has limited clinical effect.

The Beijing Haoxi biotechnology limited company finds that the learning and memory capacity of the FAD transgenic mice treated by transferrin is obviously improved. Transferrin has the main physiological function of transporting iron ions, so that hemoglobin has oxygen supply capacity, is natural human plasma protein and is safe within a physiological concentration range. The mechanism of action in this animal model is not well defined.

Disclosure of Invention

In view of the complexity of the pathogenesis of AD, one of the objects of the present invention is to provide the use of a combination composition of transferrin and mannolite sodium capsules. The combined use of the transferrin and the mannolite sodium capsules can obviously improve the cognitive level effect of patients with mild cognitive dysfunction. The invention proves the effect of the combination of the FAD transgenic AD model, the scopolamine induced dementia animal model and the Lipopolysaccharide (LPS) induced cognitive dysfunction animal model by carrying out in vivo drug effect experiments.

The invention also aims to provide application of transferrin, which can obviously improve the cognitive level of mild cognitive dysfunction.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the present invention provides the use of transferrin in the manufacture of a medicament for the prevention or/and amelioration or/and treatment of mild cognitive impairment as a result of reduced neurotransmitter or neuronal damage caused by causes other than increased abeta, and which meets clinical diagnostic criteria for cognitive levels associated with mild cognitive impairment.

In the use of the first aspect of the present invention, as a preferred embodiment, the mild cognitive dysfunction is mild cognitive dysfunction caused by brain trauma or neuroinflammation, or mild cognitive dysfunction that meets clinical diagnostic criteria without clear causes.

In a second aspect, the invention provides the use of a transferrin in combination with mannolite sodium capsules in the manufacture of a medicament for the prevention or/and amelioration or/and treatment of mild cognitive impairment.

In the use of the second aspect of the present invention, as a preferred embodiment, the mild cognitive dysfunction is mild cognitive dysfunction caused by brain trauma or neuroinflammation, or mild cognitive dysfunction diagnosed according to clinical criteria without clear causes.

In the use of the second aspect of the present invention, as a preferred embodiment, the mannolite sodium capsule is replaced with a dosage form other than a capsule containing the same pharmaceutically active ingredient as the mannolite sodium capsule.

In the application of the second aspect of the invention, as a preferred embodiment, when the transferrin is used in combination with the mannolite sodium capsules, the effective dose of the mannolite sodium capsules is 50-100mg/kg, and the effective dose of the transferrin is 5-15 mg/kg.

Compared with the prior art, the invention has the following beneficial effects:

the inventors found that transferrin has an improving effect on cognitive level reduction not caused by decrease in neurotransmitters or neuronal damage due to increase in abeta by performing experiments on an animal model of scopolamine-induced dementia and an animal model of Lipopolysaccharide (LPS) -induced cognitive dysfunction, and thus demonstrated that transferrin is suitable for improving cognitive level reduction caused by various causes, such as mild cognitive dysfunction caused by vascular diseases, mild cognitive dysfunction caused by frontotemporal atrophy, mild cognitive dysfunction caused by brain trauma, cognitive dysfunction caused by neuroinflammation, and the like.

In addition, in the present invention, through experiments, the inventors also found that: the transferrin is used together with the manna sodium capsule, so that the effect of the manna sodium capsule on improving mild cognitive dysfunction is further improved.

Drawings

FIG. 1: in the detection example 1, a water maze behavioural experiment detects the memory improvement condition of mice treated by the mannolite sodium capsule, the transferrin + mannolite sodium capsule, the transferrin, donepezil and the transferrin + donepezil, and each group of mice has a stage-up time in a hidden platform training stage; wherein, the abscissa is: training days (trainingday), ordinate: escape latency (escapelatiency);

FIG. 2: in the detection example 1, a water maze behavioural experiment detects the memory improvement condition of mice treated by the mannolite sodium capsule, the transferrin + mannolite sodium capsule, the transferrin, donepezil and the transferrin + donepezil, and the percentage of time of each group of mice in a target quadrant in a test stage; wherein, the ordinate is: percentage of time the target quadrant is located (timestent target quadrant);

FIG. 3: in the water maze behavioural experiment in detection example 2, each group of mice is subjected to the stage-loading time in the hidden platform training stage, wherein the abscissa is as follows: training days (trainingday), ordinate: escape latency (escapelatiency).

FIG. 4: in the water maze behavioural experiment in detection example 2, the percentage of time that each group of mice is located in the target quadrant in the test stage is as follows, wherein the ordinate is: the target quadrant is in percent of time (timestent target quadrant).

FIG. 5: in the water maze behavioural experiment in detection example 3, the percentage of time that each group of mice is located in the target quadrant in the test stage is as follows, wherein the ordinate is: maze arm selection times were varied as a percentage of total maze arm entry times (Alternation%).

Wherein, represents p < 0.05; represents p < 0.01; the straight line segment at the upper part in the figure represents that statistical analysis is carried out between the groups under the corresponding two end points of the line segment.

Detailed Description

The following examples further illustrate the present invention in detail, and the scope of the present invention includes, but is not limited to, the following examples.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

The materials, methods, and examples herein are illustrative only and, unless otherwise specified, should not be construed as limiting. Only suitable methods and materials are described herein, and methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

The mild cognitive dysfunction of the present invention, which is not specifically described, means that the cognitive level of a patient is decreased due to any definite reason or no definite reason, and meets the clinical diagnosis standard for the cognitive level of the mild cognitive dysfunction. Mild cognitive dysfunction is a transitional state between normal and dementia.

The transferrin used in the present invention can be a commercially available product or can be extracted from the CohnIV fraction of human plasma according to the following method, which allows for waste utilization. Preferably, the purity of the ferritin is more than 95 wt%.

The mannolite sodium capsule used in the present invention may be replaced by any dosage form other than a capsule containing the same pharmaceutically active ingredient as the mannolite sodium capsule (i.e., mannolite sodium, chemical name oligo-beta-1 → 4-D-mannuronic acid disodium). Dosage forms other than the capsule include those in which only sodium mannolite is present.

The preparation method of the transferrin comprises the following steps:

the method sequentially comprises the following steps: preparing a pretreatment solution, purifying by hydrophobic chromatography, and concentrating and displacing.

Step one, preparation of pretreatment liquid: the purpose of this step is to reconstitute the proteins in the human plasma CohnIV fraction precipitate, remove the filter aid, and remove some of the contaminating proteins after reconstitution.

Step one, first redissolution treatment: and dissolving the CohnIV component solid into the PBS buffer solution, and uniformly mixing to obtain a first compound solution.

The mass-to-volume ratio (w/v) of the human plasma CohnIV component to the PBS buffer solution is 1: (2-10), preferably 1: 3.5; the PBS buffer solution is: 10-50mmol/LPBS, pH6.0-8.0, preferably 20mmol/LPBS, pH 7.5.

Step two, first centrifugal treatment: centrifuging the first redissolution, and retaining the supernatant as the first supernatant.

The rotation speed of the centrifugation is 4000-15000rpm, the temperature is 0-10 ℃, preferably 10000rpm, and the temperature is 4 ℃.

Step three, first filtration treatment: filtering the first supernatant with 0.1-1.0um, preferably 0.45 μm filter membrane to obtain filtrate as first filtrate.

Step four, pH adjustment treatment: adding 1mol/L sodium citrate solution into the first filtrate to obtain filtrate with pH value adjusted to 6.5-7.5, preferably 7.0.

Step five, impurity protein removal treatment: stirring the filtrate after adjusting the pH value, and slowly adding a protein precipitator into the filtrate until the precipitate automatically settles and becomes solid (the solid means that the supernatant and the precipitate have obvious boundary lines, the supernatant becomes transparent, and the precipitate amount does not increase any more), and then taking the supernatant as impurity-removed protein solution for next centrifugation.

The protein precipitant is preferably polyethylene glycol 4000(PEG4000), and the mass volume ratio of PEG4000 to the filtrate after pH adjustment is 10-20%, preferably 15%.

The protein precipitant can also be ethanol solution with volume percentage concentration of 90-98%, preferably 96%, temperature of-10 deg.C, preferably-4 deg.C; the ethanol solution is kept at a low temperature because heat is generated during the precipitation process by using the ethanol solution.

The method can effectively remove a part of impurity protein, and can precipitate viruses, thereby improving the safety of the product.

Step six, second centrifugal treatment: centrifuging the protein solution without impurities, and keeping the supernatant as a second supernatant.

Step seven, second filtering treatment: filtering the second supernatant with 0.1-1.0 μm, preferably 0.45 μm filter membrane to obtain filtrate as human plasma CohnIV fraction pretreatment solution.

Step two, hydrophobic chromatography purification: the aim of the step is to remove other protein impurities from the pretreatment liquid of the human plasma CohnIV component and separate and obtain the purified transferrin.

Step one, buffer solution replacement treatment:

in this step, a HiTrap desaling Desalting column was used to replace the pretreated Cohn IV fraction from human plasma, and the initial salt concentration and pH were adjusted to the pH of the pretreated Cohn IV fraction in an equilibrium buffer (10-50mmol/L PBS, pH6.0-8.0, 0.5-3mol/L (NH)₄)₂SO₄Preferably 20mmol/L PBS, pH7.0, 1.5mol/L (NH)₄)₂SO₄) The same is true. The specific operation is as follows: (1) the HiTrap desaling Desalting column is pretreated with 5-10 column volumes of equilibration buffer (10-50mmol/L PBS, pH6.0-8.0, wherein (NH)₄)₂SO₄PBS at a concentration of 0.5-3mol/L, preferably 20mmol/L, pH7.0, wherein (NH)₄)₂SO₄Concentration of 1.5mol/L) until the conductivity detector indicates that the pH of the effluent liquid is consistent with that of the equilibration buffer. (2) An appropriate amount of the above-mentioned human plasma CohnIV fraction pretreatment solution was then fed to a hitrap purification column. (3) Further adding an equilibration buffer (including 10-50mmol/L PBS, pH6.0-8.0, wherein (NH)₄)₂SO₄PBS at a concentration of 0.5-3mol/L, preferably 20mmol/L, pH7.0, wherein (NH)₄)₂SO₄Concentration of 1.5mol/L) was eluted to the HiTrap desaling column, and the protein solution was collected according to the UV280 peak pattern to obtain a Cohn IV protein solution as a replacement buffer.

Step two, hydrophobic chromatography treatment:

in this fractionation step, an Octyl hydrophobic column (Octyl Sepharose 4Fast Flow packing) was used.

The specific operation is as follows: (1) pre-treating with 5-10 column volumes of equilibration buffer (10-50mmol/LPBS, pH6.0-8.0, wherein (NH)₄)₂SO₄A concentration of 1.5-3mol/L, preferably 20mmol/L PBS, pH7.0, wherein (NH)₄)₂SO₄Concentration 1.5mol/L) of the octyl hydrophobic column until the conductivity detector shows that the pH of the effluent liquid is consistent with that of the equilibration buffer. (2) An appropriate amount of the Cohn IV protein solution of the above displacement buffer was then fed to an octyl hydrophobic column. (3) Then adding equilibration buffer (10-50mmol/L PBS, pH6.0)-8.0, wherein (NH)₄)₂SO₄A concentration of 1.5-3mol/L, preferably 20mmol/L PBS, pH7.0, wherein (NH)₄)₂SO₄Concentration of 1.5mol/L) was performed, the target component was sufficiently bound to the octyl hydrophobic column, and the flow-through liquid (also called transliquid) was collected according to the UV280 peak pattern. (4) Then eluting with elution buffer (10-50mmol/L PBS, pH6.0-8.0, wherein (NH)₄)₂SO₄PBS at a concentration of 0.9-1.4mol/L, preferably 20mmol/L, pH7.0, wherein (NH)₄)₂SO₄Concentration of 1.2mol/L) is carried out, and the obtained eluent is the purified transferrin.

Step three, concentration and replacement:

step one, concentration treatment: concentrating the purified transferrin with 1KD-10KD, preferably 3KD ultrafiltration membrane to obtain concentrated solution. The chromatographic process of the second step can dilute the sample, and the aim of the present separation step is to concentrate the sample to a usable degree.

Step two, replacement treatment: and (2) replacing high-concentration salt (mainly ammonium sulfate) in the concentrated solution into the replacement buffer solution by adopting a method of dialysis or ultrafiltration by using the replacement buffer solution, and taking a protein solution in a dialysis membrane or a protein solution on an ultrafiltration membrane, namely the transferrin. The aim of this fractionation step is to displace the buffer containing high concentrations of salts (mainly ammonium sulfate) in the concentrate into the PBS buffer.

Wherein the used replacement buffer is 10-50mmol/L PBS, pH7.0-7.4, preferably 20mmol/L PBS, pH7.2.

When dialysis is adopted: preferably, the replacement buffer is used as dialysis buffer, the dialysis time is 20-72h, preferably 24h, and the dialysis volume ratio is 1: (100-10000), preferably 1: 500, the protein solution in the dialysis membrane is retained, namely the transferrin.

When ultrafiltration is used: preferably, the above replacement buffer solution is used as ultrafiltration buffer solution, the specification of the ultrafiltration membrane is 1-10KD, preferably 3KD, the pressure is 0.1-0.3MPa, preferably 0.1MPa, and the protein solution on the ultrafiltration membrane is retained, i.e. transferrin.

The mass-to-volume ratio (w/v) referred to in the present invention means: the ratio between the mass of the solid (g) and the volume of liquid (ml), such as: the mass-to-volume ratio (w/v) of the human plasma Cohn fraction to the PBS buffer was 1: 3.5, it means that when 1g of human plasma Cohn IV fraction was measured, 3.5ml of PBS buffer was measured.

Through comparison experiments, the in vivo drug effect of transferrin prepared from blood plasma by Beijing Haoxisi Biotechnology Limited does not have obvious difference with the in vivo drug effect of human plasma transferrin purchased from sigma company. Transferrin (TF) adopted in the experiment is purchased by sigma company, and the goods number is as follows: t3309, which is a partially saturated transferrin. The following experimental examples and examples were performed for tail vein injection using: TF solution, its preparation method is as follows: 5-15mg TF was dissolved in 5ml PBS to prepare a 1-3mg/ml solution, wherein 20mmol/L PBS, pH7.2 was used.

The mannitol sodium capsule (Jiu phase one) adopted in the embodiment of the invention is a commercially available product and is produced by Shanghai green grain pharmacy Co. The amount of the mannolite sodium capsules mentioned in the following experimental examples and examples was substantially the content of the capsules, and GV-971 for intragastric administration to mice was obtained by diluting and dissolving the content of the capsules, and was prepared as follows: 50-100mg of the active ingredient of the GV-971 content is dissolved in 10ml of PBS to prepare a solution with a concentration of 5-10mg/ml, wherein the PBS used is 20mmol/L PBS, pH 7.2.

Donepezil (DPZ) used in the examples of the present invention is a commercially available product, manufactured by drug industry ltd. DPZ for gastric lavage is prepared by dissolving 50-100mg DPZ effective component in 10ml PBS to obtain 5-10mg/ml solution, wherein the PBS is 20mmol/L PBS, pH 7.2.

The scopolamine solution for intraperitoneal injection in the embodiment of the invention is prepared by dissolving commercially available scopolamine (sigma) in physiological saline to prepare a solution with the concentration of 0.4 mg/mL.

In the embodiment of the invention, LPS for intraperitoneal injection is prepared by dissolving commercially available LPS into physiological saline to prepare a solution with the concentration of 0.4 mg/mL.

Detection example 1

The drug effect of transferrin and transferrin combined with mannot sodium capsules is verified on a 10-month-old 5xFAD transgenic animal model.

(1) The mice were divided into seven groups of 16 mice each.

First group (WT-PBS): wild Type (WT) mice were used as controls, and mice were gavaged with PBS (20. mu.l/g based on body weight); PBS was injected into the tail vein (5. mu.l/g based on body weight). This group was blank control;

second group (TG-PBS): 5 × FAD transgenic mice, each mouse gavage PBS (20 μ l/g based on body weight); PBS was injected into the tail vein (5. mu.l/g based on body weight). This group was the model control;

third group (TG-GV-971): 5XFAD transgenic mice, each mouse gavage GV-971 effective component (100mg/kg body weight) and tail vein injection PBS (5 μ l/g calculated according to body weight);

fourth group (TG-TF + GV-971): 5XFAD transgenic mice, each mouse gavage GV-971 effective component (100mg/kg body weight) and tail vein injection TF (15mg/kg body weight);

fifth group (TG-DPZ): 5XFAD transgenic mice, each mouse gavage DPZ effective component (100mg/kg body weight) and tail vein injection PBS (5 μ l/g calculated according to body weight);

sixth group (TG-TF + DPZ): 5XFAD transgenic mice, each mouse gavage DPZ effective component (100mg/kg body weight) and tail vein injection TF (15mg/kg body weight);

seventh group (TG-TF): 5XFAD transgenic mice, each mouse tail vein injection TF (15mg/kg body weight);

remarking: WT is a wild type mouse; TG is 5x FAD transgenic mice; PBS is phosphate buffer solution; TF is transferrin; GV-971 is a mannitol sodium capsule; DPZ is donepezil.

The administration of the 7 groups of the medicaments by intragastric administration is once a day, and the injection of the tail vein is once every three days.

The Morris water maze training was performed 30 days after gavage.

(2) The length of the incubation period recorded in the water maze experiment determines the spatial discrimination ability of the mice. The water maze device consists of a circular water pool with the diameter of 120cm and the height of 50cm and a movable platform with the diameter of 10cm, a camera is arranged above the water pool and is connected with an automatic computer recorder, and the water temperature is kept at (22 +/-3) DEG C.

(3) The training on the 1 st to 5 th days is hidden platform training, the platform is placed at a fixed position and is 1cm lower than the water surface, titanium dioxide is added to make the platform invisible, the heads of mice are gently and randomly placed towards the wall of the pool into one of 4 quadrants in the pool, each mouse swims for 60s in each test, finds the platform in the water and stays for 5 s; if the mice failed to find a platform after entering the water, the time was recorded as 60s and manually placed on the platform for 20s, with each mouse repeating the test 4 times a day.

(4) And (3) withdrawing the platform 24h after the last positioning navigation experiment is finished, putting the mouse into the movement track of the underwater tracker for 60s, testing the movement track and the latency of the mouse in 60s, and taking the number of times of passing through the position quadrant of the original placed platform and the time of passing through the position quadrant of the original placed platform as space memory forming indexes, and performing comparison among groups.

The test results are shown in fig. 1 and fig. 2, wherein fig. 1 shows the time of the mouse to stage in the hidden platform training phase, and fig. 2 shows the time percentage of the mouse in the target quadrant in the test phase.

In fig. 1, in the hidden platform training stage, the WT-PBS group and TG-PBS group learning curves (escape latency) are significantly different from each other at

days

2 and 3, and tend to be consistent later, and the TG-TF + GV-971 and TG-TF + DPZ group learning curves are similar to the WT + PBS group (escape latency). All mice learned to remember the location of the hidden platform prior to testing.

In fig. 2, the percentage of time that each group is located in the target quadrant is as follows: first group (WT-PBS): 32.3 + -7.8, a second group (TG-PBS) 22.5 + -6.7 (p <0.01vs. WT-PBS), a third group (TG-GV-971) 25.3 + -3.9 (p >0.05vs. TG-PBS), a fourth group (TG-TF + GV-971) 32.7 + -3.1 (p <0.01vs. TG-PBS; p <0.05vs. TG-GV971, TG-TF + DPZ, TG-TF), a fifth group (TG-DPZ) 22.2 + -3.9 (p >0.05vs. PBS), a sixth group (TG-TF + DPZ) 27.4 + -2.1 (p >0.05vs. TG-PBS; p <0.05vs. TG-DPZ; p <0.05vs. TG-971), a seventh group (TG-TF): 27.6 + -2.3 (p <0.05vs. TG-TF + GV971, TG-DPZ; p >0.05vs. TG-TF + DPZ), it can be seen that GV-971, or donepezil, injected alone, did not significantly increase the time of the mouse in the target quadrant for the Alzheimer's model mouse. Although TF alone increased the time in the target quadrant significantly compared to the TG-PBS group, the time in the target quadrant significantly increased after the combined use of GV-971 and transferrin compared to TF alone; the combination of donepezil and transferrin can not obviously improve the target quadrant time of mice, namely the effect of the combination of transferrin and GV-971 on the aspect of treating the Alzheimer disease is better than that of transferrin and donepezil, the effect of GV-971 on the aspect of treating the Alzheimer disease is better than that of TF on the aspect of treating the Alzheimer disease.

The results show that the effect of combining GV-971 and transferrin on improving the cognitive level of mice in an Alzheimer's disease model is better than the effect of singly injecting GV-971 and better than the effect of singly injecting TF;

compared with the combination of donepezil and transferrin, the injection of transferrin can improve the cognitive level of GV-971 on mice in Alzheimer's disease model more obviously.

Detection example 2

The drug effect of transferrin and transferrin combined with mannitol sodium capsules is verified on an scopolamine-induced dementia animal model.

(1) The C57BL/6 mice were divided into the following seven groups of 16 mice each.

Group one (Saline-PBS): blank control wild type mice (i.e., healthy mice), mice gavage with PBS (20. mu.l/g based on body weight); PBS was injected into the tail vein (5. mu.l/g based on body weight). This group was blank control;

second set (Scop-PBS): scopolamine-induced mouse dementia model, each mouse was gavaged with PBS (20 μ l/g based on body weight); PBS was injected into the tail vein (5. mu.l/g based on body weight). This group was the model control;

third group (Scop-GV-971): in a scopolamine-induced mouse dementia model, each mouse is perfused with GV-971 active ingredients (100mg/kg body weight) and tail vein injection PBS (5 mul/g calculated according to body weight);

fourth group (Scop-TF + GV-971): in a scopolamine-induced mouse dementia model, each mouse is perfused with GV-971 active ingredients (100mg/kg body weight) and injected with TF (15mg/kg body weight) via tail vein;

fifth group (Scop-DPZ): in a scopolamine-induced mouse dementia model, the effective component (100mg/kg body weight) of each mouse is gavaged with DPZ and PBS (5 mul/g is calculated according to the body weight) is injected into tail vein;

sixth group (Scop-TF + DPZ): in a scopolamine-induced mouse dementia model, the effective components (100mg/kg body weight) of each mouse are gavaged with DPZ and TF (15mg/kg body weight) is injected into tail veins;

seventh group (Scop-TF): scopolamine-induced mouse dementia model, each mouse was injected caudal vein with TF (15mg/kg body weight);

remarking: saline is a control mouse injected with normal Saline intraperitoneally; scop is a mouse dementia model induced by intraperitoneal injection of scopolamine; PBS is phosphate buffer solution; TF is transferrin; GV-971 is a mannitol sodium capsule; DPZ is donepezil. The scopolamine-induced mouse dementia model induces dementia symptoms by decreasing neurotransmitters (acetylcholine) in mouse brain using scopolamine.

The administration of the above 7 groups by intragastric administration and tail vein injection was once a day.

Morris water maze training was performed 7 days after gastric and tail vein injections. The water maze training and the positioning sailing experiment are the same as the 5x FAD animal model embodiment. The difference is that: before the water maze training on the 1 st to 5 th days and half an hour before the first training on each day, 2mg/kg (the administration volume is 5ml/kg) of scopolamine is respectively injected into the abdominal cavity of the mice of the second to seventh groups according to the weight of the mice to induce learning and memory impairment, so as to manufacture the mouse dementia models of each group. At the same time, the control group (first group) was injected with normal saline (5ml/kg) intraperitoneally.

The test results are shown in fig. 3 and fig. 4, wherein fig. 3 shows the time for the mouse to stage in the hidden platform training phase, and fig. 4 shows the time percentage for the mouse to stay in the target quadrant in the test phase.

In fig. 3, in the hidden platform training stage, differences between the Saline-PBS group and the Scop-PBS group learning curves (escape latency) at

days

2, 3, 4 and 5 are significant, which indicates that scopolamine successfully induces learning and memory defects of mice. Scop-TF + GV-971, Scop-TF + DPZ and Scop-TF omics learning curves are similar to those of a salt + PBS group (escape latency), which indicates that the learning capacity of the mice is improved by the three administration modes. All mice learned to remember the location of the hidden platform prior to testing.

In fig. 4, the percentage of time that each group is located in the target quadrant is, in order: group one (Saline-PBS): 38.6 + -7.5, second group (Scop-PBS) 20.3 + -5.6 (p <0.01vs. salt-PBS), third group (Scop-GV-971) 26.3 + -6.9 (p >0.05vs. Scop-PBS), fourth group (Scop-TF + GV-971) 38.1 + -6.1 (p <0.01vs. Scop-PBS, Scop-GV-971, Scop-DPZ, Scop-TF; p <0.05vs. Scop-TF + DPZ), fifth group (Scop-DPZ) 27.2 + -6.7 (p <0.05vs. Scop-PBS; p <0.01vs. Scop-TF + GV-971), sixth group (Scop-TF + DPZ) 30.4 + -5.9 (p <0.01 v.s-PBS; p <0.05 v.s-TF + GV-971; Scop-TF + Scop-0.05 v-PBS), seventh group (Scop-TF + GV-971): 27.3 ± 5.5((p <0.05vs. scop-PBS; p <0.01vs. TG-TF + GV971), it can be seen that for the scopolamine-induced cognitive dysfunction model mouse, the single injection of GV-971 can not significantly improve the time of the mouse in the target quadrant, the single injection of TF can significantly improve the time of the mouse in the target quadrant, after the GV-971 and transferrin are combined, the significance of the improvement of the mice in the target quadrant time is higher; TF or donepezil can significantly increase the time of the mouse in the target quadrant, meanwhile, the effect of the combination of the transferrin and the GV-971 on the aspect of treating the cognitive dysfunction is obviously better than the effect of the combination of the transferrin and the donepezil, the effect of the combination of the GV-971 on the aspect of treating the cognitive dysfunction is better than the effect of the combination of the GV-971 on the aspect of treating the cognitive dysfunction, and the effect of the combination of the transferrin and the GV-971 on the aspect of treating the cognitive dysfunction is better than the effect of the combination of TF on the aspect of treating the cognitive dysfunction.

The results show that the effect of combining GV-971 and transferrin on improving the cognitive level of scopolamine-induced cognitive dysfunction model mice is superior to the effect of singly injecting GV-971 and the effect of singly injecting TF;

Detection example 3

The pharmacodynamic action of transferrin and transferrin combined with mannot sodium capsules is verified on a cognitive dysfunction animal model induced by Lipopolysaccharide (LPS), wherein the model belongs to a cognitive dysfunction model caused by neuroinflammation.

second group (LPS-PBS): LPS-induced cognitive dysfunction model in mice, each mouse was gavaged with PBS (20 μ l/g based on body weight); PBS was injected into the tail vein (5. mu.l/g based on body weight). This group was the model control;

third group (LPS-GV-971): LPS-induced mouse cognitive dysfunction model, each mouse gavage GV-971 effective component (100mg/kg body weight) and tail vein injection PBS (5 μ l/g calculated according to body weight);

fourth group (LPS-TF + GV-971): mouse cognitive dysfunction model induced by LPS, each mouse gavage GV-971 effective component (100mg/kg body weight) and tail vein injection TF (15mg/kg body weight);

fifth group (LPS-DPZ): LPS-induced mouse cognitive dysfunction model, wherein each mouse is perfused with DPZ effective components (100mg/kg body weight) and injected with PBS (5 mul/g calculated according to body weight) via tail vein;

sixth group (LPS-TF + DPZ): LPS-induced mouse cognitive dysfunction model, wherein each mouse is perfused with DPZ effective components (100mg/kg body weight) and injected with TF (15mg/kg body weight) via tail vein;

seventh group (LPS-TF): LPS-induced cognitive dysfunction model in mice, each mouse was injected with TF (15mg/kg body weight) via tail vein;

remarking: saline is a control mouse injected with normal Saline intraperitoneally; LPS is a mouse cognitive dysfunction model induced by intraperitoneal injection of LPS; PBS is phosphate buffer solution; TF is transferrin; GV-971 is a mannitol sodium capsule; DPZ is donepezil. The model of cognitive dysfunction in mice induced by LPS was the symptom of cognitive dysfunction caused by neuroinflammation induced by intraperitoneal injection of LPS (0.3 mg/kg of animal body weight) for 5 consecutive days before administration.

The administration of the above 7 groups by intragastric administration and tail vein injection was once a day. The Y maze experiment was performed 7 days after gastric lavage and tail vein injection.

(2) The Y maze consists of 3 maze arms. The periphery of the Y-shaped labyrinth is shielded by a curtain, and a visual indication is pasted on the bracket. The laboratory room was dimly lit to increase mouse exploration, and the lighting above the center of the Y maze was indirect homogeneous (150-. Mice were placed in the Y maze starting at the end of one of the fixed maze arms, facing towards the center of the maze, and allowed to explore freely for 8 minutes in the Y maze. The sequence and number of times the mouse entered 3 maze arms of the Y maze were recorded (the mouse's hindpaw entered the maze arm, calculated as the arm entered the maze arm). If the mouse jumps out of the Y maze, the mouse is quickly placed back into the Y maze. After each test, the Y maze was wiped with 75% alcohol, air dried and then tested again. Mice were analyzed for the change in maze arm selection as a percentage of total maze arm entry (Alternation%). The mice naturally like exploration, and the reduction of the selection percentage indicates that the memory function of the experimental mice is reduced. The results in fig. 5 show that both TF alone and TF in combination with GV-971 or DPZ were significantly improved in alternation, with no significant difference between the three, compared to the model group 2. Whereas GV-971 or DPZ alone increased alternance% but not significantly. It can be seen that TF injection alone and GV-971 or DPZ in combination improve cognitive decline caused by neuroinflammation.

Claims

1. Use of transferrin for the manufacture of a medicament for the prevention or/and amelioration or/and treatment of mild cognitive impairment as a result of reduced cognitive levels as a result of reduced neurotransmitters or neuronal damage caused by causes other than increased abeta, in accordance with clinical diagnostic criteria for cognitive levels in mild cognitive impairment.

2. The use according to claim 1, wherein the mild cognitive dysfunction is mild cognitive dysfunction caused by brain trauma or neuroinflammation, or mild cognitive dysfunction that meets clinical diagnostic criteria without clear causes.

3. The application of the transferrin and mannatide sodium capsule in preparing the medicine for preventing or/and improving or/and treating mild cognitive dysfunction.

4. The use according to claim 3, wherein the mild cognitive dysfunction is mild cognitive dysfunction due to brain trauma or neuroinflammation, or mild cognitive dysfunction diagnosed according to clinical criteria without a clear cause.

5. Use according to claim 3 or 4, wherein the Mantidic sodium capsules are replaced by dosage forms other than capsules containing the same pharmaceutically active ingredient as the Mantidic sodium capsules.

6. The use according to claim 5, wherein the effective dose of the capsule of natritrin is 50-100mg/kg and the effective dose of transferrin is 5-15mg/kg when transferrin is administered in combination with the capsule of natritrin.