PHARMACEUTICAL COMPOSITION, COMPLEMENTARY KIT AND APPLICATION THEREOF

Abstract

A pharmaceutical composition, a complementary kit and an application thereof; the pharmaceutical composition comprises pramipexole and safinamide, the mass ratio of pramipexole to safinamide being 1:300-1:30; and the pharmaceutical composition has a good curative effect and has few side effects, and effectively improve the condition of a patient. The capabilities thereof in restoring dopamine levels are better than those from using pramipexole or safinamide alone. In addition, the composition has better neuroprotective activity, and therefore damage to the dopaminergic system and the normal sensitivity of the dopamine system to dopamine agonists are reduced. The composition can restore or balance a potential unbalanced response to “increasing dopaminergic treatment” that is caused by the use of a dopamine agonist alone; the composition can reduce the amount of pramipexole when used alone so as to reduce the risk of side effects and drug resistance.

Claims

1. A pharmaceutical composition comprising pramipexole and safinamide, wherein a mass ratio of the pramipexole and the safinamide is 1:300 to 1:30.

2. The pharmaceutical composition according to claim 1, wherein the mass ratio of the pramipexole and the safinamide is 1:200 to 1:50.

3. The pharmaceutical composition according to claim 1, wherein an amount of the pramipexole is 0.01 to 5 mg; or an amount of the safinamide is 0.3 to 1500 mg.

4. The pharmaceutical composition according to claim 3, wherein the amount of the pramipexole is 0.02, 0.04, 0.05, 0.06, 0.08, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3 or 4 mg; or the amount of the safinamide is 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 12, 14, 15, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300.0 or 1400 mg.

5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition comprises 0.05 mg of pramipexole and 7.5 mg of safinamide, 0.05 mg of pramipexole and 10 mg of safinamide, 0.05 mg of pramipexole and 15 mg of safinamide, 0.1 mg of pramipexole and 15 mg of safinamide, 0.15 mg of pramipexole and 15 mg of safinamide, 0.2 mg of pramipexole and 15 mg of safinamide, 0.5 mg of pramipexole and 15 mg of safinamide, 0.6 mg of pramipexole and 60 mg of safinamide, 1.2 mg of pramipexole and 120 mg of safinamide, 0.3 mg of pramipexole and 30 mg of safinamide, or 0.2 mg of pramipexole and 20 mg of safinamide.

6. The pharmaceutical composition according to claim 1, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

7. A kit of parts comprising a kit A and a kit B, wherein the kit A comprises pramipexole and the kit B comprises safinamide, or the kit A comprises pramipexole and safinamide and the kit B comprises another drug; wherein a mass ratio of pramipexole and safinamide is 1:300 to 1:30.

8. The kit of parts according to claim 7, wherein an amount of the pramipexole is 0.01 to 5 mg or an amount of the safinamide is 0.3 to 1500 mg.

9. (canceled)

10. A method of improving movement function or increasing dopamine concentration in a subject in need thereof, comprising administrating the pharmaceutical composition according to claim 1.

11. A method of treating Parkinson's disease comprising: administering to a subject in need thereof the pharmaceutical composition according to claim 1.

12. The pharmaceutical composition according to claim 1, wherein the mass ratio of the pramipexole and the safinamide is 1:150 to 1:75.

13. The pharmaceutical composition according to claim 1, wherein the mass ratio of the pramipexole and the safinamide is 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180 or 1:190.

14. The pharmaceutical composition according to claim 3, wherein the amount of the pramipexole is 0.05 to 0.5 mg; or the amount of the safinamide is 7.5 to 100 mg.

15. The kit of parts according to claim 7, wherein the mass ratio of pramipexole and safinamide is 1:200 to 1:50.

16. The kit of parts according to claim 15, wherein the mass ratio of pramipexole and safinamide is 150 to 1:75.

17. The kit of parts according to claim 16, wherein the mass ratio of pramipexole and safinamide is 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180 or 1:190.

18. The kit of parts according to claim 8, wherein the amount of the pramipexole is 0.05 to 0.5 mg; or the amount of the safinamide is 7.5 to 100 mg.

19. The kit of parts according to claim 18, wherein the amount of the pramipexole is 0.02, 0.04, 0.05, 0.06, 0.08, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3 or 4 mg, or the amount of the safinamide is 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 12, 14, 15, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 or 1400 mg.

20. The kit of parts according to claim 19, wherein the kit of parts comprises 0.05 mg of pramipexole and 7.5 mg of safinamide, 0.05 mg of pramipexole and 10 mg of safinamide, 0.05 mg of pramipexole and 15 mg of safinamide, 0.1 mg of pramipexole and 15 mg of safinamide, 0.15 mg of pramipexole and 15 mg of safinamide, 0.2 mg of pramipexole and 15 mg of safinamide, 0.5 mg of pramipexole and 15 mg of safinamide, 0.6 mg of pramipexole and 60 mg of safinamide, 1.2 mg of pramipexole and 120 mg of safinamide, 0.3 mg of pramipexole and 30 mg of safinamide, or 0.2 mg of pramipexole and 20 mg of safinamide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 illustrates the average dopamine concentration in mouse brain obtained by measuring the amount of mouse striatal dopamine in Example 1.

[0040] FIG. 2 illustrates the trend of APO-induced circling behavior in 6-OHDA--induced rat PD model.

[0041] FIG. 3 illustrates the revolutions of APO-induced circling in 6-OHDA-induced rat PD model on Day 29 post-dose.

DETAILED DESCRIPTION

[0042] The present disclosure is further illustrated by the following examples, which are not intended to limit the present disclosure. Experimental procedures without specified conditions in the following examples were conducted in accordance with conventional procedures and conditions, or in accordance with the manufacturer's manual.

EXAMPLE 1

In vivo characterization of the combination in an MPTP(1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine)-induced mouse Parkinson's disease model

[0043] (1) Materials

[0044] Animals: male C57BL/6J mice (SPF grade), from Nanjing Biomedical Research Institute of Nanjing University, qualification number: 201900586. The mice were fed in a closed environment at 20 to 26° C. and a relative humidity of 40% to 70?/ in a 14/10-h light/dark cycle, and were provided with free access to water and food.

[0045] Compounds:

[0046] Pramipexole (compound A), batch No.: BPL20171102, molecular weight: 302.26, purity: 99.93%, Shanghai Boyier Chemical Co., Ltd

[0047] Safinami de (compound B), batch No.: G059-S-2-151102, molecular weight: 398.45, purity: 99.97%, manufactured by Shanghai Pharmaceuticals Holding Co., Ltd.

[0048] MPTP, batch No.: N1012A, molecular weight: 209,71, purity: 98% (for induction of Parkinson's disease model), purchased from Meilunbio.

[0049] (2) Methodology

[0050] In this experiment, male C57BL/6 mice aged around 8-10 weeks were intraperitoneally injected with MPTP (20 mg/kg) twice a week (Monday and Thursday every week) for five weeks; test compounds A and B were intraperitoneally injected once daily (QP) for 6 consecutive weeks, and were given 30 min before MPTP on days of MPTP administration. Animals in control group were injected with an equal volume of noi mat saline. See Table 1 below for grouping. After 6 weeks, mice were euthanized and the dorsal striatum was separated after normal saline perfusion, weighed and preserved in a −80° C. freezer. The content of dopamine in dorsal striatal was determined by LC-MS.

TABLE-US-00001 TABLE 1 Treatment of MPTP-induced mouse Parkinson's disease model with the combination or monotherapies Number of animals Grouping (n) MPTP treatment Test compound treatment: Group 1 n = 5 0 mg/kg, intraperitoneal 0 mg/kg, intraperitoneal (G1) injection, twice a injection, QD, for 6 weeks week, for 5 weeks Group 2 n = 12 20 mg/kg, intraperitoneal 0 mg/kg, intraperitoneal (G2) injection, twice a injection, QD, week, for 5 weeks for 6 weeks Group 3 n = 12 20 mg/kg, intraperitoneal Compound B, 15 mg/kg, (G3) injection, twice a week, intraperitoneal injection, for 5 weeks QD, for 6 weeks Group 4 n = 12 20 mg/kg, intraperitoneal Compound A, 0.05 mg/kg (G4) injection, twice a week, intraperitoneal injection, for 5 weeks QD, for 6 weeks Group 5 n = 12 20 mg/kg, intraperitoneal Compound A, 0.1 mg/kg, (G5) injection, twice a week, intraperitoneal injection, for 5 weeks QD, for 6 weeks Group 6 n = 12 20 mg/kg, intraperitoneal Compound A, 0.15 mg/kg (G6) injection, twice a week, intraperitoneal injection, for 5 weeks QD, for 6 weeks Group 7 n = 12 20 mg/kg, intraperitoneal Compound A, 0.2 mg/kg, (G7) injection, twice a week, intraperitoneal injection, for 5 weeks QD, for 6 weeks Group 8 n = 12 20 mg/kg, intraperitoneal Compound A, 0.5 mg/kg, (G8) injection, twice a week, intraperitoneal injection, for 5 weeks QD, for 6 weeks Group 9 n = 12 20 mg/kg, intraperitoneal Compound A (0.05 mg/kg, (G9) injection, twice a week, QD) + compound B for 5 weeks (15 mg/kg, QD) intraperitoneal injection, for 6 weeks Group 10 n = 12 20 mg/kg, intraperitoneal Compound A (0.1 mg/kg, (G10) injection, twice a week, QD) + compound B for 5 weeks (15 mg/kg, QD), intraperitoneal injection, for 6 weeks Group 11 n = 12 20 mg/kg, intraperitoneal Compound A (0.15 mg/kg, (G11) injection, twice a week, QD) + compound B for 5 weeks (15 mg/kg, QD), intraperitoneal injection, for 6 weeks Group 12 n = 12 20 mg/kg, intraperitoneal Compound A (0.2 mg/kg, (G12) injection, twice a week, QD) + compound B for 5 weeks (15 mg/kg, QD), intraperitoneal injection, for 6 weeks Group 13 n = 12 20 mg/kg, intraperitoneal Compound A (0.5 mg/kg, (G13) injection, twice a week, QD) + compound B for 5 weeks (15 mg/kg, QD), intraperitoneal injection, for 6 weeks Note: the administration volume was 10 mL/kg. On day one, the test compounds were first injected intraperitoneally and then MPTP was injected intraperitoneally 30 min later. For animals in groups 9 to 13, MPTP was intraperitoneally administered 30 min after administration of compounds A and B.

[0051] (3) Results

[0052] The P values for comparisons of the above groups with the normal saline group G1, the modeling group G2 and the compound B group G3 are shown in Table 2 below (T-TEST unpaired t test, two-tailed):

TABLE-US-00002 TABLE 2 T-TEST- T-TEST- T-TEST- G1 G2 G3 (Normal (Modeling B Group saline) group) (15 mg/kg) Group 1 Normal VS Saline 0.00015867 0.004128274 (G1) saline group Group 2 Modeling 0.00015867  VS Model 1.33956E−10 (G2) group Group 3 Compound B 0.004128274 1.33956E−10 VSB (G3) (15 mg/kg) Group 4 Compound A 0.000108676 0.000560427 4.33604E−09 (G4) (0.05 mg/kg) Group 5 Compound A 0.000414224 0.001230318 0.000124081 (G5) (0.1 mg/kg) Group 6 Compound A 0.000181923 0.000448433 3.37881E-07 (G6) (0.15 mg/kg) Group 7 Compound A 0.001307066 0.013642873 0.012684014 (G7) (0.2 mg/kg) Group 8 Compound A 0.001764261 0.001936585 0.035856788 (G8) (0.5 mg/kg) Group 9 Compound A 0.038747195 3.39672E−08 0.02117314 (G9) (0.05 mg/kg) + Compound B (15 mg/kg) Group 10 Compound A 0.873635498 0.000230077 0.010381237 (G10) (0.1 mg/kg) + Compound B (15 mg/kg) Group 11 Compound A 0.125276833 7.07842E−11 3.31108E−05 (G11) (0.15 mg/kg) + Compound B (15 mg/kg) Group 12 Compound A 0.954019053 4.63272E−05 0.003097193 (G12) (0.2 mg/kg) + Compound B (15 mg/kg) Group 13 Compound A 0.247059784 3.65199E−05 0.025978093 (G13) (0.5 mg/kg) + Compound B (15 mg/kg)

[0053] (1) Comparison with normal saline group Gl:

[0054] Compared with G1, groups G2, G4, G5 and G6 showed that P<0.001, suggesting extremely significant differences in striatal dopamine concentration; compared with Gi, groups G3, G7 and G8 showed that 0.001 <P<0.01, suggesting significant differences; compared with G1, group G9 showed that 0.01 <P<0.05, suggesting a difference; compared with G1, groups G10, Gil, G12 and G13 showed that P >0.05, suggesting no differences.

[0055] (2) Comparison with modeling group G2:

[0056] Compared with G2, group G7 showed that 0.001<P<0.01, suggesting a significant difference in striatal dopamine concentration; compared with G2, group G8 showed that P 0.05, suggesting no difference; compared with G2, group G13 showed that 0.01<P<0.05, suggesting a difference; compared with G2, the other groups showed that P<0.001, suggesting extremely significant differences.

[0057] (3) Comparison with compound B group G3:

[0058] Compared with G3, group G7 showed that P>0.05, suggesting no difference in striatal dopamine concentration; compared with G3, groups G8, G9 and G13 showed that 0.01<P<0.05, suggesting significant differences; compared with G3, the other groups showed that P<0.001, suggesting extremely significant differences.

[0059] The above results show that the dopamine levels in dorsal striatum in the modeling group G2 were significantly lower than those in the normal saline group G1; except for group G8, the dopamine levels in dorsal striatum in the compound A groups were significantly higher than those in the modeling group G2, while group G8 showed no significant difference; the dopamine levels in dorsal striatum in the compound B groups were significantly higher than those in the modeling group G2; the dopamine levels in dorsal striatum in the combination. groups were significantly higher than those in the modeling group G2, and showed no significant difference from those in the normal saline group GI. This indicates that, after treated with the combination therapy, the dopamine levels significantly increased in dorsal striatum in MPTP-modeled mice; the dopamine levels of the compound A (0.1 mg/kg) +compound B (15 mg/kg) group GIO and the compound A (0.2 mg/kg) +compound B (15 mg/kg) group G12 demonstrated the least significant differences from the normal saline group G1 and the most significant differences from the modeling group G2, suggesting the best efficacies.

[0060] The corresponding results are shown in FIG. I. As shown in the figure, pramipexole and safinamide both demonstrated improvement effects on dopamine reduction in this model, and the combination of the two drugs is significantly elevated the improvement effects. The pramipexole injection monotherapy showed dose-dependent effect to a certain extent; for the combination, although the dose dependence was irregular due to greater errors in several groups and an optimal combination cannot be determined, it can be seen that after the second dose group (G10), the efficacy was not improved significantly and the highest dose group even showed a significant reduction. This indicated that a good efficacy can be achieved at a low dose of pramipexole in the combination, which is consistent with the final purpose of reducing the dose of pramipexole so as to reduce risks of side effects and drug resistance while achieving similar or superior therapeutic effect. From this result, we can determine a range of the combination where pramipexole (0,05, 0.1, 0.15, 0.2 or 0.5 mg/kg) has a synergic effect with safinamide (15 mg/kg). Considering that the amount of pramipexole used is minimized without affecting the efficacy (and thus the side effects can be reduced), it is believed that the optimal effect of the experiment can be achieved when 0.1 mg/kg of pramipexole is combined with 15 mg/kg of safinamide, i.e., the dopamine concentration in the brain tissue of the animal model can be maximized.

EXAMPLE 2

In vivo characterization of the combination in a rat Parkinson's disease model with 6-OHDA (6-hydroxydopamine)-induced unilateral damage

[0061] (1) Materials

[0062] 150 Wistar rats, male, supplier: Beijing YR. The animals were sent to Shanghai Ruizi Chemical Research Co., Ltd., and accommodated in the animal room at 2-3 mice/cage at 20-26° C. in a 12/12-h light/dark cycle (5:00 am to 5:00 pm in light).

[0063] Pramipexole hydrochloride: Manufacturer: Adamas; Purity: 99%; batch No.: P1448882

[0064] Safinamide mesylate: Manufacturer: Shanghai Phan laceuticals Holding Co., Ltd.; Purity: 99.95%; batch No.: G059-S-2-151104

[0065] 6-1-1ydroxydopamine: Manufacturer: Sigma Aldrich (Shanghai) Trading Co., Ltd.; batch No.: MKCD0817.

[0066] Other primary reagents and compounds are shown in Table 3 below:

TABLE-US-00003 TABLE 3 Specifi- Batch Date of Name Manufacturer cations No. expiration Sodium Merck 25 NA 2020 Jan. 31 pentobarbitone g/bottle Ascorbic acid National Institutes 100 mg/ 100425- NA for Food and Drug vial 201504 Control, PRC Apomorphine USP 250mg R08440 NA

[0067] (2) Formulation

[0068] 2.1 Formulation of 0.5 mg/kg Apo solution

[0069] 2,1.1 4.44 mg of apomorphine (CF=1.17) was weighed accurately and added to 15.180 mL of sterile normal saline. The mixture was vortexed for 10 min in dark to obtain a clear and transparent solution. The solution was prepared right before use, and was kept in ice in use.

[0070] 2.2 Formulation of 6-OHDA

[0071] 2.2.1 0.02% (0.02 g/100 mL) ascorbic acid: 7.81 mg of ascorbic acid was weighed accurately and added to 39.05 mL of normal saline. The mixture was vortexed for 2 min to obtain a clear solution. The formulation process was conducted in dark and the solution was preserved at 4° C.

[0072] 2,2.1 5 mg/mL 6-OHDA (0.02% ascorbic acid): 0.663 mL of 0.02% ascorbic acid was added to the vial of 6-0FIDA (5 mg package) (freebase=98%×5 mg/1.479=3.313 mg). The mixture was vortexed for 2 min to obtain a clear solution. A second vial was prepared using the same method. The 5 μg/μL 6-01-1DA solution was subpackaged into 33 vials of 40-μL stock solution, which were preserved at −80° C. The entire process was conducted in dark.

[0073] 2.3 Formulation of 15 mg/kg (3 mg/mL) safinamide mesylate

[0074] 2.3.1 359.83 mg of safinamide mesylate (CF=1,318, 99.95% purity) was weighed accurately, and added to 90.959 mL of ultrapure water. The mixture was vortexed for 5 min in dark to obtain a clear and transparent solution, which was preserved at 4 degrees.

[0075] 2.4 Formulation of 0.45 mg/kg (0.9 mg/mL) pramipexole hydrochloride

[0076] 2.4.1 6.74 mg of pramipexole hydrochloride (CF=1.430, 99% purity) was weighed accurately, and added to 51.844 mL of ultrapure water. The mixture was vortexed for 5 min in dark to obtain a clear and transparent solution, which was preserved at 4 degrees.

[0077] 2.5 Formulation of 0.1 mg/kg (0.02 mg/mL) pramipexole hydrochloride

[0078] 2.5,1 2.87 mg of pramipexole hydrochloride (CF=1.430, 99% purity) was weighed accurately, and added to 99.350 mL of ultrapure water. The mixture was vortexed for 5 min in dark to obtain a clear and transparent solution, which was preserved at 4 degrees.

[0079] All formulations were conducted based on this standard and were diluted proportionally.

[0080] (3) Procedures

[0081] 3.1 The animals were pre-screened by an apomot le-induced contralateral circling test before surgery.

[0082] 3.1,1 On the day of testing, the animals were adapted in the operation room for more than 30 min.

[0083] 3.1.2 The animals were injected intraperitoneally with APO (0.5 mg/kg, 2 mL/kg) 3 minutes before testing.

[0084] 3.1.3 The animals were placed in a circular test box after dosing. Changes in animal behavior were observed and recorded by Ethovi si on video software, and the number of revolutions of each animal within 30 min was counted manually.

[0085] 3.1.4 APO-induced circling behavior: The animal circled rightwards with the left hind limb as the fulcrum. One turn was counted as one revolution by taking the head of the rat as a reference. Animals with more than 180 revolutions within 30 min were qualified rat Parkinson's disease models.

[0086] 3.1.5 After the test, the animals were returned to the homecage.

[0087] 3.2 Preparation of 6-OHDA-induced rat Parkinson's disease model

[0088] 3.2.1 140 animals were randomly selected, and intramuscularly injected with Zoletil at 30 mg/kg in combination with 3 mg/kg of xylazine hydrochloride for general anesthesia before the operation.

[0089] 3,2.2 Rats were fixed on a stereotaxic apparatus. The skull epidermis was incised, the anterior and posterior fontanels were found, and the tooth bar was adjusted to keep the skull surface horizontal.

[0090] 3.2.3 Taking the anterior fontanel as zero point, the coordinates were identified on the skull surface: AP: 0.5 mm; ML: −2.8 mm; DV: −4.5 mm.

[0091] 3.2.4 A round hole slightly larger than the diameter of the micro injection needle was drilled at the coordinates by using a skull drill. The micro injection needle was adjusted to the surface of the dura mater and set as the zero point.

[0092] 3.2.5 The light was turned off and a yellow light source was turned on. A volume of 6-OHDA slightly greater than the injection amount was taken in.

[0093] 3.2.6 The micro injection needle was slowly screwed down to a designated depth in the brain of the rat within about 5 min.

[0094] 3.2.7 The injection of 6-OHDA was started 2 min after the needle reached the designated depth. The injection amount was 4 μL (5 μg/μL), with the total amount of 6-OHDA being 20 .sub.jig. The injection speed was 1 μL/min.

[0095] 3.2.8 After the injection was finished, the needle was left for 5 min. The micro injection needle was then slowly screwed out in about 5 min.

[0096] 3.2.9 After the wound was sutured and disinfected, the rat was returned to the homecage. The rats were kept warm and water was given before the rats regained their consciousness. The wound, respiration and pain frequency, and excretion were observed.

[0097] 3.2.10 The surgical instruments were cleaned and the micro injector was rinsed with normal saline to prevent clogging everyday after operation.

[0098] 3.2.11 The animals were subjected to a 3-day postoperative care.

[0099] 3.3 Qualified animal models were screened on Days 7, 14 and 21 after operation by an apomorphine-induced contralateral circling test in the PD rats.

[0100] 3.3.1 On the day of testing, the animals were adapted in the operation room for more than 30 min.

[0101] 3.3.2 The animals were injected intraperitoneally with APO (0.5 mg/kg, 2 mL/kg) 3 minutes before testing.

[0102] 3.3.3 The animals were placed in a circular test box after dosing. Changes in animal behavior were observed and recorded by Ethovision video software, and the number of revolutions of each animal within 30 min was counted manually.

[0103] 3.3.4 APO-induced circling behavior: The animal circled rightwards with the left hind limb as the fulcrum. One turn was counted as one revolution by taking the head of the rat as a reference. Animals with more than 180 revolutions within 30 min were qualified rat Parkinson's disease models.

[0104] 3.3.5 After the test, the animals were returned to the homecage,

[0105] 3,4 Therapeutic effect of continuous treatment on 6-OHDA-induced rat Parkinson's disease model

[0106] 3.4,1 Apomorphine-induced contralateral circling test was performed on day 21 after operation, 78 animals with >180 revolutions in 30 min and 10 naïve animals, for a total of 88 animals, were selected for administration and testing.

[0107] 3.4.2 Qualified rats were assigned to various groups based on the number of revolutions on day 21 after operation to ensure similar levels of lesions among the groups before treatment.

[0108] 3.4.3 After grouping, the treatment was given on day 22 after operation. The grouping is shown in Table 4 below.

TABLE-US-00004 TABLE 4 Number Route of Frequency of admin- of admin- Group Model Treatment animals istration istration G1 Wild type Normal saline 10 P.O. QD × 28 G2 6-OHDA- Normal saline 16 P.O. QD × 28 induced (included) G3 unilateral Safinamide 16 P.O. QD × 28 damage PD SAF (15 mpk) (included) G4 model Low-dose 16 P.O. QD × 28 (Male wistar pramipexole (included) rat) PPX (0.1 mpk) G5 Combination 15 P.O. QD × 28 (PPX 0.1 (included) mpk/SAF 15 mpk) G6 High-dose 15 P.O. QD × 28 pramipexole (included) PPX (0.45 mg/kg)

[0109] In the table, PPX denotes pramipexole, and SAF denotes safinamide.

[0110] 3.4.4 Apomorphine-induced circling test was performed prior to dosing on days 7, 14, 21 and 28.

[0111] 3.4.5 The rats were subjected to the apomorphine-induced circling test the day after the last dose.

[0112] (4) Results

[0113] Results of the apomorphine-induced circling test pre-dose, on days 7, 14, 21 and 28 and the next day of the last dose are shown in FIG. 2.

[0114] Compared with the control group (vehicle group), the combination group G5 (PPX 0.1 mpkISAF 15 mpk) showed improved movement function of a rat PD model with timeliness and significance after 21 days of treatment, and a better efficacy in improving the movement function in the rat PD model than two monotherapy groups (G3 and G4); the combination also imparts neuroprotective activity and thus can reduce the normal sensitivity of the dopamine system to dopamine agonists, reducing or avoiding fluctuations in symptoms. After 29 days of treatment, the combination group G5 showed a 36% reduction in the number of revolutions in the rat PD model compared with that before treatment, while groups G3 and C14 showed a 5% elevation and a 24% reduction, respectively (see FIG. 3).

[0115] It can also be seen from the above data that the composition of this example can restore or equilibrate the potential inequilibrium response to the dopaminergic increase treatment caused by dopamine agonist alone; the composition reduces the amount of pramipexole to reduce the risks of side effects and drug resistance, while achieving the same or better therapeutic effect.

[0116] Although specific embodiments of the present disclosure have been described above, it will be appreciated by those skilled in the art that these embodiments are merely illustrative and that many changes or modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure. The scope of protection of the present disclosure is therefore defined by the appended claims.