USE OF FULLERENE STRUCTURE IN PREPARATION OF MEDICAMENTS FOR TREATING PARKINSON'S DISEASE
20210046107 ยท 2021-02-18
Inventors
- Chunru Wang (Beijing, CN)
- Mingming Zhen (Beijing, CN)
- Xue Li (Beijing, CN)
- Hui Li (Beijing, CN)
- Xuekai Bai (Beijing, CN)
- Chunli Bai (Beijing, CN)
Cpc classification
International classification
A61K47/44
HUMAN NECESSITIES
A61K9/14
HUMAN NECESSITIES
Abstract
An application of a fullerene structure in the preparation of medications for treating Parkinson's disease. The fullerene structure comprises at least one of the following active ingredient groups: a fullerene, a metallofullerene, and a composition of the fullerene and the metallofullerene; an oil-soluble fullerene, an oil-soluble metallofullerene, and a composition of the oil-soluble fullerene and the oil-soluble metallofullerene; a water-soluble fullerene, a water-soluble metallofullerene, and a composition of the water-soluble fullerene and the water-soluble metal-lofullerene; the medicinal esters of the nine elements, or the medicinal salts of the nine elements.
Claims
1. A method of treating Parkinson's disease, comprising the step of: administering an effective dose of a fullerene structure to a subject in need of treatment for Parkinson's disease, wherein the fullerene structure comprises oil-soluble C.sub.60 or oil-soluble C.sub.70.
2. The method according to claim 1, wherein the oil-soluble C.sub.60 is obtained through oil-soluble modification of C.sub.60, and the oil-soluble C.sub.70 is obtained through oil-soluble modification of C.sub.70.
3. The method according to claim 2, wherein the oil-soluble modification of C.sub.60 disperses the C.sub.60 in the oil solution to obtain an oil-soluble modified liquid.
4. The method according to claim 2, the oil-soluble modification of C.sub.60 comprises the step of coating the C.sub.60 with a first oil solution, and the oil-soluble modification of C.sub.70 comprises the step of coating the C.sub.70 with a second oil solution.
5. The method according to claim 4, wherein the first oil solution or the second oil solution is a single ingredient oil or a mixture of different oils, and the oil is one or more of vegetable oil or animal fat.
6. The method according to claim 5, wherein the oil is selected from olive oil, linseed oil, sunflower oil, corn germ oil, soybean oil, or squalane.
7. The method according to claim 6; wherein the dispersing comprises: ball milling or ultrasonication of the mixture of the C.sub.60 and the oil solution, followed by centrifugation to remove the precipitate, and filtration of the resulting supernatant.
8. The method according to claim 6, wherein a concentration of the C.sub.60 in the oil-soluble C.sub.60 is from 0.01 to 100 mg/mL.
9. The method according to claim 6, wherein a concentration of the C60 in the oil-soluble C60 is from 0.4 to 0.8 mg/mL.
10. The method according to claim 2, wherein the oil-soluble modification of C.sub.70 disperses the C.sub.70 in the oil solution to obtain an oil-soluble modified liquid.
11. The method according to claim 10, wherein the dispersing comprises: ball milling or ultrasonication of the mixture of the C.sub.70 and the oil solution, followed by centrifugation to remove the precipitate, and filtration of the resulting supernatant.
12. The method according to claim 10, wherein a concentration of the C.sub.70 in the oil-soluble C.sub.70 is from 0.01 to 100 mg/mL.
13. The method according to claim 10, wherein a concentration of the C.sub.70 in the oil-soluble C.sub.70 is from 0.4 to 0.8 mg/mL
14. The method according to claim 1, wherein the Parkinson's disease includes physical disabilities and dyskinesia caused by Parkinson's disease, including limbs tremor, rigid limbs, hypokinesia, bradykinesia, or discordant movement.
15. The method according to claim 1, wherein the fullerene structure is applied at a dose of from 0.01 mg/kg/d to 1000 mg/kg/d.
16. The method according to claim 1, wherein the fullerene structure is applied at a dose of from 0.1 to 10 mg/kg/d, or 4 mg/kg/d.
17. The method according to claim 1, wherein the C.sub.60 or the C.sub.70 of fullerene structure is applied at a dose of from 0.5655 mg/kg/d to 1.131 mg/kg/d.
18. The method according to claim 17, wherein the fullerene structure is administered orally, by injection or intraperitoneally.
19. The method according to claim 1, wherein the subject is a human, a mouse, a guinea pig, a rat, a dog, a rabbit, or a monkey.
20. The method according to claim 1, wherein the fullerene structure comprises C.sub.60 coated with olive oil.
Description
DESCRIPTION OF THE DRAWINGS
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DESCRIPTION OF THE PREFERRED EMBODIMENT
[0079] The present disclosure will be described below with reference to specific embodiments through the accompanying drawings, but the present disclosure is not limited to the following embodiments.
[0080] Solid powders of the fullerene body C.sub.60 and C.sub.70 and solid powders of the metallofullerene body Gd@C.sub.82 with a purity of 99% used in the examples of the present disclosure were all purchased from Xiamen Funano New Material Technology Company LTD. Other materials, reagents, and instruments used can be commercially available unless otherwise specified; other experimental methods are all conventional methods unless otherwise specified.
EXAMPLE 1
Preparation of Oil-Coated Hollow Fullerenes and/or Oil-Coated Metallofullerenes
[0081] 20 ml of olive oil was measured, 20 mg of C.sub.60 or 20 mg of C.sub.70 or 20 mg of Gd@C.sub.82 (particle sizes are all 0.7-1 nm) was weighed, and they were mixed and stirred evenly to obtain a mixture; then, the mixture was placed in a ball mill to be ball-milled for 10 hours, after that, the mixture was taken out, stored in a cool, dry and dark place, and left standstill for 1 hour, the precipitate was centrifuged to be removed, and then the resulting supernatant was filtered using a 220 nm filter membrane to get final product. In the present disclosure, the oil-soluble fullerene prepared from the olive oil and C.sub.60 according to the above method was called C.sub.60-olive oil for short, wherein the content of the C.sub.60 was 0.8 mg/mL; the oil-soluble fullerene prepared from the olive oil and C.sub.70 according to the above method was called C.sub.70-olive oil for short; the oil-soluble metallofullerene prepared from the olive oil and Gd@C.sub.82 according to the above method was called Gd@C.sub.82-olive oil for short, wherein the content of the Gd@C.sub.82 was 0.4 mg/mL.
EXAMPLE 2
Preparation of Water-Soluble Hydroxylated Hollow Fullerenes and/or Water-Soluble Hydroxylated Metallofullerenes
[0082] 1) 100 mg of Gd@C.sub.82 solid powder or 100 mg of C.sub.60 solid powder was added to a 100 ml of single-mouth flask, 7 ml of a 30% by volume aqueous hydrogen peroxide solution and 3 ml of a 2M aqueous sodium hydroxide solution were respectively added, and the oil bath was heated to 70 C. to be reacted for 2-5 h;
[0083] 2) after the reaction, small molecules were removed by an M.W.=3500 dialysis bag, and a conductivity meter would be used for monitoring until the dialysis was completed. A water-soluble hydroxylated gadolinium metallofullerene or a water-soluble hydroxylated fullerene can be obtained from the product resulted from concentration. Upon DLS test, the average particle sizes of the water-soluble hydroxylated gadolinium metallofullerenes in aqueous solution were 140 nm, and the particle size distribution was uniform; the average particle size of the water-soluble hydroxylated fullerene in the aqueous solution was 200 nm, and the particle size distribution was uniform.
EXAMPLE 3
Test of Free Radicals-Scavenging Ability of the Fullerenes
[0084] The free radicals-scavenging ability of the oil-soluble fullerenes and oil-soluble metallofullerenes was detected by electron spin resonance spectrum (ESR).
[0085] The method of producing hydroxyl radical by UV induction was adopted for testing. 50 L of hydrogen peroxide with 39% mass concentration, 50 L of PBS buffer solution (pH 7.4), and a small amount (0.133 mM) of lutidine N-oxide (DMPO, free radical trapping agent) were mixed. The blank control group was directly irradiated with 280 nm UV for 4 min, and the experimental group was immediately added 10 L, of Gd@C.sub.82-olive oil or C.sub.60-olive oil prepared according to Example 1 and irradiated with 280 nm of UV light for 4 min, and the radical signals were detected. ESR data was obtained, as shown in
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[0087]
EXAMPLE 4
Cell Experiment
[0088] Commonly used Hela cells were taken as a cell experimental model to verify that the Gd@C.sub.82-olive oil and C.sub.60-olive oil obtained in Example 1 have the ability to scavenge free radicals and protect cells.
[0089] (1) Medicament Toxicity Experiment
[0090] Hela cells were cultured in high-glucose DMEM medium containing 10% of serum, trypsinized and subcultured to a sufficient number and then were used to carried out the medicament toxicity experiment.
[0091] In the cell suspension obtained by trypsinization, the cell density was 510.sup.4/mL. 200 L of cell suspension was added to each well of a 96-well plate and incubated for 24 h. The medium was aspirated, and 10% serum-containing high-glucose DMEM medium as a blank medium and Gd@C.sub.82-olive oil medium of different concentrations were added respectively in different wells (Gd@C.sub.82-olive oil medium of different concentrations refers to adding different amounts of the Gd@C.sub.82-olive oil obtained in Example 1 in a blank medium to form Gd@C.sub.82-olive oil medium of different concentrations). The concentrations of the Gd@C.sub.82-olive oil in Gd@C.sub.82-olive oil medium of different concentrations were 5, 10, 20, 50 and 100 M respectively. The mixtures in different wells were incubate for 24 h, then the blank medium or Gd@C.sub.82-olive oil medium were aspirated, the remaining cells were washed with PBS for three times and 100 L, of colorless DMED and 10 L of CCK-8 were added to each well, the mixture in each well was incubated for 1 h and the cell activity was tested with a microplate reader. The results are as shown in
[0092] In accordance with the same experimental procedures described above, the Gd@C.sub.82-olive oil was replaced with C.sub.60-olive oil for experiment. The results of the cell activity are as shown in
[0093] (2) Free Radicals Scavenging Experiment
[0094] Hela cells were cultured in high-glucose DMEM medium containing 10% of serum, trypsinized and subcultured to a sufficient number and then carried out free radicals scavenging experiment. In the cell suspension obtained by trypsinization, the cell density was 510.sup.4/mL. 200 L of cell suspension was added to each well of a 96-well plate and incubated for 24 h, and the medium was aspirated. High-glucose DMEM medium containing 10% of serum was added in 16 random wells as a blank medium, H.sub.2O.sub.2 solution with a concentration of 150 M was added to each of the remaining wells and incubated for 1 h, and the H.sub.2O.sub.2 solution was aspirated. Gd@C.sub.82-olive oil medium of different concentrations were added in different wells (Gd@C.sub.82-olive oil medium of different concentrations refers to adding different amounts of the Gd@C.sub.82-olive oil obtained in Example 1 into a blank medium to form Gd@C.sub.82-olive oil medium of different concentrations). The concentrations of the Gd@C.sub.82-olive oil in Gd@C.sub.82-olive oil medium of different concentrations were 0, 10, 20, 40 and 50 M respectively. The mixtures in different wells were incubate for 3 h and then the blank medium or Gd@C.sub.82-olive oil medium were aspirated, the remaining cells were washed with PBS for three times and 100 L, of colorless DMED and 10 L, of CCK-8 were added to each well, the mixture in each well was incubate for 1 h and the cell activity was tested with a microplate reader. The results are as shown in
[0095] In accordance with the same experimental steps described above, Gd@C.sub.82-olive oil was replaced with C.sub.60-olive oil for the experiment. The results are as shown in
EXAMPLE 5
Animal Behavioral Experiment Part 1
[0096] (1) The Establishment of Parkinson's Disease Model
[0097] Many studies have shown that there are various factors for the pathogenesis of Parkinson's disease, and neurotoxins play an important role in the pathogenetic process of Parkinson's disease. MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride) was the earliest neurotoxin discovered associated with Parkinson's disease. MPTP entered the brain, and was first taken up by glial cells, then converted to MPP+ under the effect of monoamine oxidase, and MPP+ was actively taken up by dopaminergic neurons, then entered the mitochondria to inhibit the activity of respiratory chain complex I, so free radicals were produced, ATPs were depleted, leading to apoptosis. A mature MPTP-induced mouse Parkinson's disease model was used to examine the efficacy of an oil-soluble fullerene structure in the treatment of Parkinson's disease.
[0098] It has been found that C57BL/6 mice are most sensitive to MPTP, so C57BL/6 mice were used by the present disclosure for experiments.
[0099] There were 40 C57BL/6 mice in present disclosure, who were 10 weeks old, from 18 to 22 g by weight, and given intraperitoneal injection of MPTP with a concentration of 35 mg/Kg, they were injected once a day for 7 days, the mice had static tremors, reduced movements, erecting tails, piloerection and so on, proving the successful establishment of the model.
[0100] (2) Mouse Therapic Experimental Regime
[0101] Among the 40 model mice of the present disclosure, 30 were taken out for experiments. Gd@C.sub.82-olive oil and C.sub.60-olive oil therapic experiments were carried out for the mice suffering from Parkinson's disease. 30 experimental mice were randomly divided into group A, group B and group C with 10 in each group. Group A was Gd@C.sub.82-olive oil experimental group, group B was C.sub.60-olive oil experimental group, and group C was control group, and the mice were intragastrically administered. Group A was intragastrically administered with 0.1 mL of Gd@C.sub.82-olive oil prepared in Example 1 per day, group B was intragastrically administered with 0.1 mL of C.sub.60-olive oil prepared in Example 1 per day, and group C was intragastrically administered with the same amount of normal saline per day. The mice were administered for the whole 10 days and observed and had the behavioral experiment test.
[0102] (3) Mouse Behavioral Experiment
[0103] The present disclosure tested the therapeutic effect of the mice by the spontaneous activity counting, suspension experiments, and swimming tests.
[0104] 1) Spontaneous Activity Count
[0105] The present disclosure refers to a conventional spontaneous activity counting method, a self-made perspex box of 30 cm30 cm15 cm was adopted, and the bottom of the box was scribed with lines into grids of 6 cm6 cm, and the testwas carried out at room temperature in a quiet and dark environment. The mice in the experimental groups and the control group were respectively tested after treatment. After the mice were adapted for 10 minutes, the number of the grids the mice had moved by and the number of times the mice had stood within 5 minutes was counted, and the average value was measured five times. The results are as shown in Table 1. As can be seen from the data in Table 1, there is a significant increase in the number of the grids the mice had moved by and the number of times the mice had stood after the mice had been treated with Gd@C.sub.82-olive oil and C.sub.60-olive oil, demonstrating that oil-soluble fullerenes or oil-soluble metallofullerenes can improve the behavior of the mice suffering from Parkinson's disease. As compared with the C.sub.60-olive oil composition, Gd@C.sub.82-olive oil composition was greater in improving the behavior of the mice suffering from Parkinson's disease.
[0106] 2) Suspension Experiment
[0107] The mice in the experimental groups and the control group were respectively detected after treatment. The mice for detection were suspended on a self-made horizontal wire. If the mice grasped the wire with 2 back claws, 3 points were scored, if the wire was grasped with 1 back claw, 2 points were scored, if no wire can be grasped, 1 point was scored, the results are as shown in Table 1. As can be seen from the data in Table 1, there was a significant improvement in the hindlimb movement and coordination ability of the mice after treatment.
[0108] 3) Swimming Experiment.
[0109] The mice were placed in a water tank of 20 cm30 cm20 cm with a temperature of from 22 to 25 C., the scoring standards are as follows: 30 points were scored for those who continuously swam within lmin. 25 points were scored for those who swam for most of the time but just occasionally floated, 20 points were scored for those who floated for more than half the time, 15 points were scored for those who swam occasionally, and 10 points were scored for those who floated on one side. The results are as shown in Table 1. From the data in Table 1, it can be seen that the experimental groups A and B have stronger swimming ability than the control group C, which indicates that the limbs movement ability has been improved.
TABLE-US-00001 TABLE 1 Mouse behavioral experiment results The number of The number of the grids the mice times the mice Suspension Swimming Groups had moved by had stood experiment experiment Experimental group A 120.21 18.21 35.20 2.98 3.01 0.59 2.98 0.45 Experimental group B 110.32 20.45 29.15 3.01 2.85 0.44 2.05 0.60 Control group C 70.11 20.34 19.01 3.31 1.58 0.74 1.49 0.54
[0110] The present disclosure mainly adopts Gd@C.sub.82-olive oil and C.sub.60-olive oil as examples, and the fullerene olive oil composition and the metallofullerene olive oil composition have been proved having strong free radicals-scavenging ability through material testing, cell level testing and animal-level experiments. The tremors and incoordination of animal limbs caused by Parkinson's disease can be effectively relieved, thus Parkinson's disease can be effectively treated.
EXAMPLE 6
Animal Behavioral Experiment Part 2
[0111] Levodopa is a common medicament for the treatment of Parkinson's disease. Its functional mechanism is to supplement the lack of dopamine in the brain. Clinically, it is often compatibly applied with the peripheral dopa decarboxylase inhibitor benserazide to improve efficacy. This experiment adopts a common clinical regime in which levodopa and benserazide were compatibly applied by 4:1 as positive control medicaments. A typical MPTP-induced mouse Parkinson's disease model was adopted by the experiment to examine the efficacy of the fullerene in the treatment of Parkinson's disease.
[0112] I. Experimental Materials
[0113] 1. Animals: the C57/BL6J mice (male, 18-20 g) were adopted for the experiment. The mice were raised in an environment of room temperature 242 C., a light-dark 12-hour cycle alternation was maintained and adequate food and clean drinking water were supplied. The mice were adopted for experiments after being raised for more than 5 days.
[0114] 2. Compounds
[0115] 2.1. Modeling medicaments
[0116] Name: MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride);
[0117] Property: off-white powder;
[0118] Product number: M0896;
[0119] Specification: 100 mg/bottle;
[0120] Storage conditions: room temperature, sealing, drying, and protection from light;
[0121] Provider: sigma.
[0122] 2.2. Positive control medicaments
[0123] Name: Levodopa (levodopa, L-DA);
[0124] Property: white powder;
[0125] Lot number: MKBQ9185V;
[0126] Specification: 5 g/bottle;
[0127] Storage conditions: room temperature, sealing, drying, and protection from light;
[0128] Provider: sigma.
[0129] Name: Benserazide;
[0130] Property: off-white powder;
[0131] Lot number: BCBN0530V;
[0132] Specification: 1 g/bottle;
[0133] Storage conditions: from 2 to 8 C., sealing, drying, and protection from light;
[0134] Provider: sigma.
[0135] 2.3. Test compounds
[0136] Name: C.sub.60-olive oil obtained in Example 1 (hereinafter abbreviated as fullerene);
[0137] Property: brown-red liquid;
[0138] Sample content: 870 mg/L.
[0139] 3. The above compound preparation method
TABLE-US-00002 Compound preparation method Name Preparation Subpackage Storage Pre-administration Modeling 4 ml of sodium 4 C., sealed and Before each medicaments chloride protected from administration, 25 MPTP injection was light. mg/ml of MPTP injected into an mother liquor was 100-mg bottle, diluted with and shaken to sodium chloride fully dissolve the injection to a 2.5 powder, with mg/ml of liquid the mother liquor for use and mixed concentration well and injected being 25 mg/ml. subcutaneously. (25 mg/kg The administration MPTP) volume was 10 ml/kg. Positive A certain amount Freshly prepared Before each medicament of L-DA powder every day. administration, levodopa (L-DA) was weighed and the medicaments sodium chloride were mixed well injection was and injected added to a final intraperitoneally. concentration of The administration 1 mg/ml. volume was 10 ml/kg. (10 mg/kg L-DA) Positive A certain amount Freshly prepared Before each medicament of benserazide every day. administration, benserazide (Be) powder was the medicaments weighed and were mixed well sodium chloride and injected injection was intraperitoneally. added to a final The administration concentration of volume was 10 ml/kg. 0.25 mg/ml. (2.5 mg/kg Benserazide) Test medicament The mother sealed and Before each (C.sub.60 - olive oil) liquor of protected from administration, fullerene with a light. the medicaments concentration of were mixed well 870 mg/L was and diluted with intragastrically olive oil to administered. The 0.2262 mg/ml, administration 0.1131 mg/ml, volume of diluted and 0.05655 liquid for use of mg/ml of liquid the fullerene was for use. 10 ml/kg.
[0140] II. Experimental Method
[0141] 1. Experimental grouping
[0142] A total of 72 C57 mice were randomly divided into 7 groups:
TABLE-US-00003 Meidcament Serial number Group name Group for short Animal number 1 Normal saline Normal saline Control group 10 control group 2 Modeling group 25 mg/kg MPTP Modeling group 11 3 Olive oil group Olive oil Olive oil group 10 4 Positive 10 mg/kg L-dopa + 11 medicaments L-dopa + Benserazide group 2.5 mg/kg group Benserazide 5 Test compound 0.5655 mg/kg 0.5655 mg/kg of 10 group fullerene test compound fullerene group 6 1.131 mg/kg 1.131 mg/kg of 10 fullerene test compound fullerene group 7 2.262 mg/kg 2.262 mg/kg of 10 fullerene test compound fullerene group
[0143] 2. Experimental regime
[0144] 2.1. Normal saline was adopted for the control group for modeling and administration, except for replacing the corresponding medicament with the normal saline, the control group's modeling and administration methods are the same as those of the other 6 groups. The other 6 groups were injected subcutaneously once a day with 25 mg/kg of MPTP for 5 days. During the modeling period, the positive medicaments or test medicament or olive oil or normal saline was also given. After modeling, the MPTP was stopped being given, the test medicament or positive medicaments or olive oil or normal saline was still given for 7 days. Administration volume was 10 ml/kg each time. The experimental flow chart for modeling and administration was as shown in
TABLE-US-00004 Treating medicament name Modeling medicament name Group and its drug-delivery way and its drug-delivery way Control group Normal saline (i.p.) Normal saline (s.c.) Modeling group Normal saline (i.p.) 25 mg/kg MPTP (s.c.) Olive oil group Olive oil (i.p.) Positive medicaments 10 mg/kg group L-dopa(i.p.) + 2.5 mg/kg Benserazi (i.p.) Test compound 0.5655 mg/kg fullerene (i.g.) group 1.131 mg/kg fullerene (i.g.) 2.262 mg/kg fullerene (i.g.)
[0145] 2.2. On the 5th, 9th and 12th days of the experiment, the behavioral test of the spontaneous activity of the mice was performed; On the 6th, 9th, and 12th days of the experiment, mice were tested for climbing poles and rotarod behaviors.
[0146] 3. Behavioral testing method
[0147] 3.1. Mouse spontaneous activity was tested by an open-field method
[0148] An open box experimental apparatus used for the open-field method was TS EMulti Conditioning systems. The length, width and height of the inner box were 45 cm45 cm33 cm respectively, and the camera recorded animal activities. In this experiment, the mice were performed spontaneous activity behavioral tests on the 5th, 9th and 12th days of the experiment. The experiment was conducted in a quiet room. The mice were placed in the center of the open box. The test lasted for 5 minutes, and the mice were observed for their activity time, activity distance, vertical activity time (mice standing time) within 5 minutes. The open box was thoroughly cleaned and the next mouse was observed.
[0149] 3.2. Bradykinesia of mice was tested by the pole test
[0150] The pole test was used to test typical behavioral symptoms in Parkinson's disease-bradykinesia. In this experiment, the mice were performed behavioral tests on the 6th, 9th, and 12th days respectively. The mouse with the head upwards was placed gently on the top of a rough pole (8 mm in diameter and 55 cm in height). The time taken by the mice from moving from the top of the rod until the head was downwards was recorded as the latent period T-turn, and the time taken by the mice from moving downward until the limbs all reached the bottom of the rod was recorded as the down-climbing time T-LA, the time over 30 s was recorded as 30 s. Each mouse was tested 5 times and an average value was obtained.
[0151] 3.3. Rotarod test was used to test the motor coordination ability of mice
[0152] The rotarod was divided into 5 horizontal sections, each was separated by a partition to ensure that the animals were not affected by each other. Rotarod speed was set to 28 rpm/2min. The mouse was placed on a 1.25 inch diameter roller and the switch was turned on. When timing started, the time taken by the mouse from starting rotating the rotarod until the mouse fell off from the roller was recorded as the latent period (i.e., the first time of falling off) as a measurement of the motor coordination ability of mice. Each mouse was tested 3 times and an average value was obtained. The mice were performed rotarod tests on the 6th, 9th, and 12th days of the experiment.
[0153] III. Experimental Results
[0154] 1. Changes in body weight of the experimental animals after MPTP modeling and medication treatment
[0155] On the second day of MPTP modeling, the body weights of the 6 groups of mice except for the normal saline control group of mice were significantly reduced, as compared with the normal saline control group of mice, the difference was significant (p<0.05-0.001); On the fourth day, weight gain was gradually recovered (see
[0156] 2. Behavioral experiment
[0157] 2.1. Mouse spontaneous activity was tested by the open-field method
[0158] On the 5th, 9th, and 12th days of the experiment, i.e., the 5th day of the modeling (i.e., 5th day of administration), the 4th day after the modeling (i.e., the 9th day of administration) and the 7th day after the modeling (i.e., the 12th day of administration), the mice's spontaneous activity was evaluated. The results were as shown in Table 2, Table 3, and Table 4 and in
TABLE-US-00005 TABLE 2 Effect of fullerene on the spontaneous activity of the model mice suffering from MPTP-induced PD (D 5) Group Activity time (s) Activity distance (s) Vertical activity time (s) Normal saline control group 152.70 9.00 (n = 10) 24.39 1.60 (n = 10) 18.27 2.33 (n = 10) Modeling group 32.22 10.54### (n = 10) .sup.4.66 1.47###(n = 10) 2.74 1.04### (n = 10) Olive oil group 33.67 5.40### (n = 10) .sup.4.81 0.72###(n = 10) 1.64 0.84### (n = 10) L-dopa + Benserazide 39.56 5.99 (n = 11) 6.06 0.87 (n = 11) 2.85 1.09 (n = 11) 0.5655 mg/kg test compound 56.33 15.97 (n = 10) 8.49 2.55 (n = 10) 5.12 4.07 (n = 10) fullerene group 1.131 mg/kg test compound 55.12 14.92 (n = 10) 8.29 2.37 (n = 10) 4.03 2.84 (n = 10) fullerene group 2.262 mg/kg test compound 62.86 11.07* (n = 10) 9.30 1.76* (n = 10) 4.30 2.20 (n = 10) fullerene group As compared with normal saline control group, ###represents p < 0.001; as compared with olive oil group, *represents p < 0.05
TABLE-US-00006 TABLE 3 Effect of fullerene on the spontaneous activity of the model mice suffering from MPTP-induced PD (D 9) Group Activity time (s) Activity distance (s) Vertical activity time (s) Normal saline control group 128.22 4.89 (n = 10) 19.95 0.83 (n = 10) 22.32 2.71 (n = 10) Modeling group 126.25 5.41 (n = 11) 19.32 0.95 (n = 11) 24.73 2.64 (n = 11) Olive oil group 120.39 7.53 (n = 10) 18.39 1.24 (n = 10) 24.87 3.50 (n = 10) L-dopa + Benserazide 109.57 5.39* (n = 11) 16.05 0.83* (n = 11) 17.82 2.02 (n = 11) 0.5655 mg/kg test compound 115.87 10.80 (n = 10) 17.70 1.78 (n = 10) 24.76 4.90 (n = 10) fullerene group 1.131 mg/kg test compound 116.57 12.20 (n = 10) 17.90 2.14 (n = 10) 21.78 4.36 (n = 10) fullerene group 2.262 mg/kg test compound 115.27 5.37 (n = 10) 17.56 1.12 (n = 10) 19.19 1.23 (n = 10) fullerene group As compared with modeling group, *represents p < 0.05
TABLE-US-00007 TABLE 4 Effect of fullerene on the spontaneous activity of the model mice suffering from MPTP-induced PD (D 12) Group Activity time (s) Activity distance (s) Vertical activity time (s) Normal saline control group 110.09 7.11 (n = 10) 16.92 1.09 (n = 10) 18.94 1.87 (n = 10) Modeling group 120.13 4.03 (n = 11) 18.31 0.77 (n = 11) 17.47 1.98 (n = 11) Olive oil group 126.07 9.85 (n = 10) 20.09 1.61 (n = 10) 26.89 4.84 (n = 10) L-dopa + Benserazide 105.11 6.36 (n = 11) 16.15 1.15 (n = 11) 16.22 2.74 (n = 11) 0.5655 mg/kg test compound 111.17 10.95 (n = 10) 17.49 1.79 (n = 10) 20.01 3.00 (n = 10) fullerene group 1.131 mg/kg test compound 123.53 7.78 (n = 10) 18.93 1.45 (n = 10) 18.16 3.49 (n = 10) fullerene group 2.262 mg/kg test compound 118.00 5.90 (n = 10) 18.08 1.10 (n = 10) 23.99 5.46 (n = 10) fullerene group
[0159] 2.2. Pole test
[0160] On the 6th, 9th, and 12th days of the experiment, the mice were tested for pole behaviors and tested for activity coordination. The results were as shown in Table 5, Table 6 and
TABLE-US-00008 TABLE 5 Effect of fullerene on latent period of pole behavior of the model mice suffering from MPTP-induced PD (S) Group 6th day 9th day 12th day Animal number (n) Normal saline control group 0.58 0.05.sup. 0.53 0.04 0.51 0.01 10 Modeling group 1.08 0.08### 0.92 0.09###.sup. 1.08 0.13### 11 Olive oil group 1.41 0.13###*.sup. 1.28 0.12###*.sup. 1.09 0.09### 10 L-dopa + Benserazide 0.46 0.02*** 0.55 0.03***.sup. 0.57 0.03*** 11 0.5655 mg/kg test compound 0.52 0.02*** 0.55 0.04** 0.88 0.06 10 fullerene group 1.131 mg/kg test compound 0.72 0.08** 0.55 0.04** 0.78 0.08.sup. 10 fullerene group 2.262 mg/kg test compound 0.51 0.03*** 0.47 0.0*** 0.71 0.06* 10 fullerene group As compared with normal saline control group, ###represents p < 0.001; as compared with modeling group, *represents p < 0.05, **represents p < 0.01, ***represents p < 0.001; as compared with olive oil group, represents p < 0.05, represents p < 0.01, represents p < 0.001
TABLE-US-00009 TABLE 6 Effect of fullerene on down-climbing time of pole behavior of the model mice suffering from MPTP-induced PD (S) Group 6th day 9th day 12th day Animal number (n) Normal saline control group 3.98 0.18 4.26 0.37.sup. 3.92 0.24 10 Modeling group 5.18 0.26## 5.32 0.33#.sup. 4.99 0.41# 11 Olive oil group 5.27 0.17### 5.66 0.23##.sup. 5.08 0.28## 10 L-dopa + Benserazide 3.78 0.25*** 3.76 0.33**.sup. 3.74 0.29* 11 0.5655 mg/kg test compound 4.43 0.20* 4.96 0.50.sup. 4.92 0.36 10 fullerene group 1.131 mg/kg test compound .sup.4.16 0.24** 4.04 0.25** 4.40 0.36 10 fullerene group 2.262 mg/kg test compound .sup.3.61 0.25*** 4.05 0.26** .sup.4.05 0.34 10 fullerene group As compared with normal saline control group, ##represents p < 0.01, ###represents p < 0.001; as compared with modeling group, *represents p < 0.05, **represents p < 0.01, ***represents p < 0.001; as compared with olive oil group, represents p < 0.05, represents p < 0.01, represents p < 0.001
[0161] 2.3. Rotarod test experiment
[0162] The experiment was conducted on the 6th, 9th, and 12th days.
[0163] The mice were subjected to the rotarod test for their motor coordination ability. The results were as shown in Table 7 and
TABLE-US-00010 TABLE 7 Effect of fullerene on latent period of rotarod behavior of the model mice suffering from MPTP-induced PD (S) Latent peroid (s) Group 6th day 9th day 12th day Animal number Normal saline control group 97.20 5.37 109.70 3.24 111.60 4.13 10 Modeling group 65.61 8.34## 70.91 9.31## 76.09 8.03## 11 Olive oil group 65.13 10.39# 77.70 9.61## 71.97 9.95## 10 L-dopa + Benserazide 96.36 7.88* 100.18 5.65* 98.09 3.65* 11 0.5655 mg/kg test compound 85.10 10.17 93.23 10.23 .sup.99.97 6.98* 10 fullerene group 1.131 mg/kg test compound 79.97 9.93 100.27 8.15* .sup.97.57 5.10* 10 fullerene group 2.262 mg/kg test compound 76.03 9.91 88.93 9.89 84.47 9.37 10 fullerene group As compared with normal saline control group, #represents p < 0.05, ##represents p < 0.01; as compared with modeling group, *represents p < 0.05; as compared with olive oil group, represents p < 0.05,
indicates data missing or illegible when filed
[0164] IV Summary of the Experiments
[0165] MPTP was injected subcutaneously for the whole 5 days for modeling, and the normal saline or olive oil or fullerene was given while modeling. Thereafter, the administration of the normal saline or olive oil or fullerene was continued for 7 days, and 12 days in all. The mice were performed behavioral evaluation on the 5th, the 6th, the 9th, and the 12th days of the administration. The results showed that:
[0166] 1. On the 5th day of experiment, the total activity time, activity distance and vertical activity time of the mice in each dose of test compound fullerene group were higher than those in the olive oil group, of which 2.262 mg/kg of test compound fullerene group was the most significant.
[0167] 2. On the 6th, 9th, and 12th days of the experiment, the pole experiment showed that all of the positive medicament group and each dose of test compound fullerene group could significantly improve the motor coordination ability of PD mice, so that the latent period and the down-climbing time were significantly shortened, and there was a statistically significant difference in those of the mice in 2.262 mg/kg of test compound fullerene group. The results showed that fullerenes can significantly relieve MPTP-induced bradykinesia and stiffness in mice and improve PD animal motor coordination ability.
[0168] 3. On the 6th, 9th, and 12th days of the experiment, the rotarod test showed that falling off from the roller for the 1.sup.st time (i.e. the latent period) of the animals in the positive medicament group and each dose of compound test fullerenegroup(i.e. latent period) was prolonged. As compared with the modeling group, there was a significant difference in the latent period of 1.131 mg/kg of test compound fullerene group (9th day of the experiment), 0.5655 mg/kg of test compound fullerene group (12th day of the experiment), and 1.131 mg/kg of the test compound fullerene group (12th day of the experiment) and the positive medicament group. On the 12th day of the experiment, there was a significant difference in the latent period of 0.5655 mg/kg and 1.131 mg/kg of test compound fullerene groups as compared with that of the olive oil group.
[0169] The experimental results showed that fullerene (i.e., C.sub.60-olive oil) can improve MPTP-induced Parkinson's disease-like symptoms in mice, and there was a certain dose-dependent relationship, and there was no obvious effect of solvent olive oil.