阅读说明：本技术 包含克拉维酸与丙戊酸的组合物及其用途 (Composition containing clavulanic acid and valproic acid and application thereof ) 是由 何应瑞 陈建宏 于 2019-11-26 设计创作，主要内容包括：一种用以治疗癫痫及/或治疗与癫痫相关的运动障碍与认知缺陷的组合物,其包含克拉维酸与丙戊酸。本发明另外还关于该组合物的用途,用以制造治疗癫痫及/或治疗与癫痫相关的运动障碍与认知缺陷的药物。(A composition for the treatment of epilepsy and/or the treatment of epilepsy-related dyskinesia and cognitive deficits comprising clavulanic acid and valproic acid. The invention further relates to the use of the composition for the manufacture of a medicament for the treatment of epilepsy and/or for the treatment of movement disorders and cognitive deficits associated with epilepsy.)

1. A composition for treating epilepsy, comprising: clavulanic acid and valproic acid.

2. A composition for treating epilepsy-related dyskinesia and cognitive deficits, comprising: clavulanic acid and valproic acid.

3. Use of a composition comprising clavulanic acid and valproic acid for the preparation of a medicament for the treatment of an epileptic condition, wherein clavulanic acid and valproic acid are co-administered to a subject in need thereof for the treatment of an epileptic seizure in said subject.

4. Use of a composition comprising clavulanic acid and valproic acid for the preparation of a medicament for the treatment of epilepsy-related dyskinesia and cognitive deficits, wherein clavulanic acid and valproic acid are co-administered to a subject in need thereof for the treatment of epilepsy-related dyskinesia and cognitive deficits in said subject.

5. The use of a composition comprising clavulanic acid and valproic acid according to claim 3 or 4, wherein clavulanic acid is administered to the subject in need thereof at an administration dose of 0.016 to 10 mg/kg/day.

6. The use of a composition comprising clavulanic acid and valproic acid according to claim 5, wherein clavulanic acid is administered to said subject in need thereof at an administration dose of 0.016 to 4.99 mg/kg/day.

7. The use of a composition comprising clavulanic acid and valproic acid according to claim 3 or 4, wherein valproic acid is administered to the subject in need thereof at an administration dose of 0.8-30 mg/kg/day.

8. The use of a composition comprising clavulanic acid and valproic acid according to claim 7, wherein valproic acid is administered to said subject in need thereof at an administration dose of 0.8-19.9 mg/kg/day.

9. Use of a composition comprising clavulanic acid and valproic acid according to claim 3 or 4 wherein clavulanic acid is clavulanic acid, a clavulanate salt derivative or a clavulanate ester derivative and valproic acid is valproic acid, a valproate derivative or a valerate ester derivative.

10. The use of a composition comprising clavulanic acid and valproic acid as claimed in claim 3 or 4, wherein clavulanic acid and valproic acid are administered to the subject in need thereof concurrently.

11. The use of a composition comprising clavulanic acid and valproic acid as claimed in claim 3 or 4, wherein clavulanic acid and valproic acid are administered to the subject in need thereof continuously.

12. The use of a composition comprising clavulanic acid and valproic acid as claimed in claim 3 or 4, wherein clavulanic acid and valproic acid are administered intermittently to the subject in need thereof.

13. The use of a composition comprising clavulanic acid and valproic acid as claimed in claim 3 or 4, wherein clavulanic acid and valproic acid are administered to the subject in need thereof sequentially.

14. The use of a composition comprising clavulanic acid and valproic acid as claimed in claim 3 or 4, wherein clavulanic acid and valproic acid are administered separately to the subject in need thereof.

15. The use of a composition comprising clavulanic acid and valproic acid as claimed in claim 3 or 4, wherein clavulanic acid is administered to the subject in need thereof orally or parenterally.

16. The use of a composition comprising clavulanic acid and valproic acid according to claim 15, where clavulanic acid is administered to the subject in need thereof intravenously, intramuscularly, intraperitoneally, transdermally, sublingually or by inhalation.

17. The use of a composition comprising clavulanic acid and valproic acid according to claim 3 or 4, wherein valproic acid is administered to the subject in need thereof orally or parenterally.

18. The use of a composition comprising clavulanic acid and valproic acid according to claim 17, wherein valproic acid is administered to the subject in need thereof by intravenous injection, intramuscular injection, intraperitoneal injection, transdermal absorption, sublingual absorption or inhalation.

Technical Field

The present invention relates to a composition comprising clavulanic acid and valproic acid, in particular for the treatment of epilepsy and/or epilepsy-related dyskinesia and cognitive deficits. The invention also relates to the use of the composition.

Background

Epilepsy (epilepsy) is a neurological physical condition characterized by seizures (also known as epileptic seizures) for a number of reasons, and abnormal electrical discharges (abnormal activity) in the brain of epileptic patients can lead to symptoms of seizures (symptoms).

Valproic acid (chemical structural formula shown in fig. 1) is the first-line drug (first-line) in the existing antiepileptic drugs (antiepileptic drugs), and can be used for preventing the symptoms of epileptic seizure, however, about 30% of patients with epilepsy cannot be effectively controlled even after being treated; furthermore, since valproic acid has side effects (side effect) such as nausea (nausea), vomiting (vomiting), sleepiness (sleepiness), dry mouth (dry mouth), and the like, and may also affect liver function (liver function) and renal tubular injury (renal tubular injury), an epileptic using valproic acid must regularly perform tests for liver function and kidney function, which causes inconvenience in life.

In addition to seizures, epilepsy can also cause nerve damage (nerve damage) or nerve cell death (nerve cell death) to the brain, causing symptoms such as dyskinesia and cognitive deficits, for example, the motor coordination and balance ability of a patient can be affected, the cognitive function and memory ability of the patient can also be affected, and the quality of life (QoL for short) of an epileptic patient can be reduced; however, the conventional antiepileptic drugs such as valproic acid cannot improve the nerve injury caused by epilepsy, and also can promote gamma-aminobutyric acid (GABA) in brain and spinal cord, wherein the gamma-aminobutyric acid is an inhibitory neurotransmitter to further inhibit nerve, behavior and cognitive function, so that the administration of the conventional antiepileptic drugs such as valproic acid cannot improve the dyskinesia and cognitive deficiency of epileptics, but can cause the worsening of the symptoms such as dyskinesia and cognitive deficiency.

In addition, the conventional antiepileptic drugs such as valproic acid must be continuously administered, and if the patient is not well compliant with the medication (medical compliance) and is not properly prescribed to take the medication (medical compliance), there is a risk of occurrence of seizures or status epilepticus, and thus, there is a need for improvement of the conventional antiepileptic drugs.

Disclosure of Invention

In order to solve the above problems, the present invention provides a composition for treating epileptic seizures, and for treating epilepsy-related dyskinesia and cognitive deficits.

It is a further object of the present invention to provide a use of a composition comprising clavulanic acid and valproic acid for the treatment of seizures, and for the treatment of epilepsy-related dyskinesias and cognitive deficits.

The composition for treating epilepsy of the present invention may comprise: clavulanic acid and valproic acid; alternatively, the compositions of the invention for treating epilepsy-related dyskinesia and cognitive deficits may comprise: clavulanic acid and valproic acid.

Therefore, the composition for treating epilepsy and the composition for treating epilepsy-related dyskinesia and cognitive deficits of the present invention can prevent epilepsy from occurring, weaken the intensity of epilepsy, and promote the regeneration of nerve cells in the region of hippocampal dentate gyrus (dentate gyrus) by the synergistic effect of clavulanic acid and valproic acid, thereby treating epilepsy and treating epilepsy-related dyskinesia and cognitive deficits (e.g., impaired status of improving motor coordination and balance ability of epileptic individuals, impaired status of object recognition ability and impaired status of memory ability) of epileptic individuals.

The composition containing the clavulanic acid and the valproic acid is used for preparing the antiepileptic medicine, wherein the clavulanic acid and the valproic acid can be co-administered to a required individual so as to treat the epileptic seizure of the required individual; alternatively, the composition comprising clavulanic acid and valproic acid of the present invention can be used for preparing a medicament for treating epilepsy-related dyskinesia and cognitive deficits, wherein clavulanic acid and valproic acid can be co-administered to a desired subject to promote the regeneration of nerve cells in the brain of the desired subject, thereby treating epilepsy-related dyskinesia and cognitive deficits in the desired subject.

Therefore, the administration dosage of the valproic acid can be reduced by co-administration with the clavulanic acid, so that metabolic organs such as liver, kidney and the like can rapidly and efficiently metabolize and decompose the valproic acid, the valproic acid can be prevented from being excessively accumulated in organisms, the nervous, mental and behavioral interference caused by the valproic acid can be reduced, the burden of the excessive administration of the valproic acid on the metabolic organs such as liver, kidney and the like can be reduced, and the aim of reducing the possibility of side effects is fulfilled.

The use of the composition of the present invention comprising clavulanic acid and valproic acid, wherein clavulanic acid can be administered to the desired subject at an administration dose of 0.016-10 mg/kg/day, preferably 0.016-4.99 g/kg/day, and valproic acid can be administered to the desired subject at an administration dose of 0.8-30 mg/kg/day, preferably 0.8-19.99 mg/kg/day; thereby, the efficacy of effectively treating the epileptic seizure of the desired individual and treating the epilepsy-related dyskinesia and cognitive deficits can be achieved.

The invention relates to an application of a composition containing clavulanic acid and valproic acid, wherein the clavulanic acid is clavulanic acid, clavulanate derivatives or clavulanate ester derivatives, and the valproic acid is valproic acid, valproate derivatives or valerate ester derivatives; thereby, the goal of effectively treating the epileptic seizure of the needed individual and treating the dyskinesia and cognitive deficit related to the epilepsia can be achieved.

The use of a composition comprising clavulanic acid and valproic acid according to the invention, wherein clavulanic acid and valproic acid may be administered to the subject in need thereof concurrently, sequentially or separately; thereby, the goal of effectively treating the epileptic seizure of the needed individual and effectively treating the dyskinesia and cognitive deficit related to the epilepsia can be achieved.

The use of the composition of the invention comprising clavulanic acid and valproic acid, wherein clavulanic acid may be administered to the subject continuously or intermittently; thereby, the goal of effectively treating the epileptic seizure of the needed individual and effectively treating the dyskinesia and cognitive deficit related to the epilepsia can be achieved.

The composition containing clavulanic acid and valproic acid is used, wherein the clavulanic acid can be orally or parenterally administered to a needed individual; for example, clavulanic acid may be administered to the subject in need thereof by intravenous injection, intramuscular injection, intraperitoneal injection, transdermal absorption, sublingual absorption or inhalation; thereby, the goal of effectively treating the epileptic seizure of the needed individual and effectively treating the dyskinesia and cognitive deficit related to the epilepsia can be achieved.

The use of the composition of the present invention comprising clavulanic acid and valproic acid, wherein valproic acid can be administered to the subject orally or parenterally; for example, valproic acid can be administered to the subject in need thereof by intravenous injection, intramuscular injection, intraperitoneal injection, transdermal absorption, sublingual absorption, or inhalation; thereby, the efficacy of effectively treating the epileptic seizure of the required individual and effectively treating the dyskinesia and cognitive deficits associated with epilepsy can be achieved.

Drawings

FIG. 1: the chemical structural formula of valproic acid;

FIG. 2: the chemical structural formula of the clavulanic acid is shown in the specification;

FIG. 3: the intensity of seizures (seizure) in rats of groups A0-A5 of trial (B);

FIG. 4 a: schematic (one) of the roller meter of test (C);

FIG. 4 b: schematic diagram (ii) of the roller meter of test (C);

FIG. 5: continuous movement time (training time) of rats of groups A0-A5 of test (C);

FIG. 6 a: schematic (one) of the open space box of test (D);

FIG. 6 b: schematic (ii) of the open space box of test (D);

FIG. 7: percentage of time explored in groups A0-A5 of rats from test (D);

FIG. 8 a: schematic representation (one) of the shuttle box of test (E);

FIG. 8 b: schematic (two) shuttle box for test (E);

FIG. 8 c: schematic (iii) shuttle box for test (E);

FIG. 8 d: schematic (iv) shuttle box for test (E);

FIG. 9: latency time for rats in groups A0-A5 of trial (E) to enter the dark compartment;

FIG. 10: the number of BrdU-positive cells in the region of the hippocampal dentate gyrus of rats in groups A0-A5 of trial (F).

Description of the reference numerals

C1 bright room

C2 dark chamber

D sliding door

O1 old article

O2 old article

O3 old article

O4 novel article

R rat to be tested

S electric shock

And a W roller.

Detailed Description

In order to make the aforementioned and other objects, features and advantages of the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below:

the clavulanic acid (clavulanic acid) has a chemical structural formula shown in fig. 2, belongs to a molecule (beta-lactam molecular) of beta-lactam, and although the effect of the clavulanic acid as an antibiotic is poor, when penicillin-group antibiotics (penicillin-group antibiotics) are combined, the antibiotic resistance (antibiotic resistance) of bacteria secreting beta-lactamase (beta-lactamase) can be overcome, otherwise most of penicillin can be inactivated.

The clavulanic acid can be administered to a desired individual to treat seizures (seizure) of the desired individual and to treat epilepsy-related dyskinesia (motor system) and cognitive impairment (cognitive impairment) and thus can be applied to the preparation of antiepileptic drugs or to the preparation of drugs for the treatment of epilepsy-related dyskinesia and cognitive impairment.

The clavulanic acid of the invention can be administered to the subject by any suitable route of administration, for example, clavulanic acid can be administered orally (orally) or parenterally (parenterally), for example, clavulanic acid can be administered to the subject by Intravenous (IV) injection, Intramuscular (IM) injection, Intraperitoneal (IP) injection, transdermal, sublingual or inhalation.

Additionally, the clavulanic acid of the invention can be administered to the desired subject at an administration dose of 0.016-10 mg/kg/day, preferably at an administration dose of 0.016-4.99 mg/kg/day lower than the clinical dose. Clavulanic acid can be administered to the subject in need thereof continuously (continuously) or intermittently (internmitently). In detail, the clavulanic acid is administered to the desired subject once every predetermined time period, in the case that the predetermined time period is not more than 24 hours, the clavulanic acid is continuously administered to the desired subject, and in the case that the predetermined time period is more than 24 hours, the clavulanic acid is intermittently administered to the desired subject. However, the dose and frequency of administration described above should be varied depending on the intended individual and the intended route of administration, and are not limited thereto.

Additionally, the clavulanic acid of the present invention can also be co-administered to the desired subject with an existing antiepileptic drug (i.e., valproic acid) such that the duration of the pharmacological effects of clavulanic acid and valproic acid can overlap with each other, thereby synergistically (synergetically) treating seizures in the desired subject and treating epilepsy-related dyskinesia and cognitive deficits in the desired subject. In particular, it means that clavulanic acid and valproic acid can be administered to the desired individual [ i.e., simultaneously clavulanic acid and valproic acid are administered to the desired individual ] in parallel (convurrently); or sequentially (sequentially) administering clavulanic acid and valproic acid to the subject [ i.e., administering clavulanic acid to the subject in preference to the subject while maintaining a therapeutically effective blood concentration of clavulanic acid (systemic drug concentration) ], i.e., administering valproic acid to the subject; for example, administration of valproic acid to the desired subject within 10 minutes to 8 hours after administration of clavulanic acid to the desired subject); or sequentially administering valproic acid and clavulanic acid to the subject in need thereof [ i.e., administering clavulanic acid to the subject in need thereof under conditions where the blood level of valproic acid remains at the therapeutically effective concentration after administering valproic acid to the subject in preference to the subject; for example, clavulanic acid is administered to the desired subject within 10 minutes to 8 hours after valproic acid is administered to the desired subject); alternatively, clavulanic acid and valproic acid may be administered separately (separately) to the desired subject [ i.e., clavulanic acid is administered to the desired subject and valproic acid is administered to the desired subject only after the blood concentration of the clavulanic acid drug is below the therapeutically effective concentration; for example, valproic acid is not administered to the desired subject until 8 to 12 hours after clavulanic acid is administered to the desired subject); alternatively, valproic acid and clavulanic acid may be administered separately to the subject in need thereof [ i.e., valproic acid is administered to the subject in need thereof and clavulanic acid is administered to the subject after the blood level of valproic acid is below the therapeutically effective concentration; for example, clavulanic acid is not administered to the desired subject until 8 to 12 hours after valproic acid is administered to the desired subject ].

Additionally, where valproic acid is co-administered with clavulanic acid of the invention to the subject in need thereof to treat seizures in the subject in need thereof, and to treat epilepsy-related dyskinesia and cognitive deficits in the subject in need thereof, valproic acid can be administered to the subject orally or parenterally, e.g., valproic acid can be administered to the subject intravenously, intramuscularly, intraperitoneally, transdermally, sublingually or by inhalation, and to the subject in need thereof at an administration dose of 0.8 to 30 mg/kg/day, preferably at an administration dose of 0.8 to 19.99 mg/kg/day lower than the clinical dose. Valproic acid can be administered to the subject continuously or intermittently. However, the dose and frequency of administration described above should be varied depending on the intended individual and the intended route of administration, and are not limited thereto.

In addition, the clavulanic acid and valproic acid of the present invention may be co-manufactured as a composition, and the clavulanic acid and valproic acid may be administered to the desired individual in parallel, sequentially or separately by adjusting the dosage form (dosage form). For example, the composition may further comprise at least one pharmaceutically acceptable carrier, such that the release profile of clavulanic acid and valproic acid in the subject in need thereof is modulated by the at least one pharmaceutically acceptable carrier; for example, clavulanic acid (or valproic acid) may be coated with liposomes (liposomes) to allow the sustained release of clavulanic acid (or valproic acid), thereby achieving sequential or separate administration of clavulanic acid and valproic acid.

To confirm that the seizure and the epilepsy-related dyskinesia and cognitive deficits can be effectively treated by the administration of clavulanic acid according to the present invention, and that the co-administration of clavulanic acid with valproic acid according to the present invention has a synergistic effect (synergistic effect) in the treatment of seizures and in the treatment of epilepsy-related dyskinesia and cognitive deficits, the following experiments were performed:

(A) induction and experimental design of epileptic rats

In this experiment, male Wistar rats (purchased from taiwan leschak biotechnology, ltd.) of 8 weeks old were kept in an animal room (the light time and the dark time are each 12 hours) maintained at room temperature of 21 to 24 ℃.

The motor function of the rats was tested by a roller test (rotarod test) on the first 3 days from the start of the test, and on the 1 st to 13 th days, pentylenetetrazol (PTZ for short, 35mg/kg per dose) was administered to the rats by intraperitoneal injection at a frequency of once every 2 days (i.e., on the 1 st, 3 th, 5 th, 7 th, 9 th, 11 th and 13 th days of the test), to obtain epileptic rats. On day 21, the intensity of seizures in each group of epileptic rats was measured after administering PTZ again by intraperitoneal injection.

Next, on the 7 th day of the experiment, clavulanic acid and/or valproic acid shown in table 1 was administered to the epileptic rats in groups a2 to a5 by intraperitoneal injection every day, and the treatment was continued for 7 days (i.e., on the 7 th to 13 th days of the experiment), and normal rats in group a0 and epileptic rats in group a1 were administered with physiological saline (1 ml/kg/day, i.p. injection).

TABLE 1 treatment conditions for the groups of rats in this experiment

In addition, on 14 th day of the experiment, the motor functions of rats to be tested in groups A0-A5 were tested by a roller tester, on 15 th-17 th day of the experiment, the damage status of the object recognition ability of rats to be tested in groups A0-A5 was tested by an object recognition test, and on 18 th-20 th day of the experiment, the damage status of the memory ability of rats to be tested in groups A0-A5 was tested by a passive avoidance test. Subsequently, on day 22 of the experiment, 5-bromo-2 ' -deoxyuridine (5 ' -bromo-2 ' -deoxyuridine, BrdU) was administered to the rats to be tested in groups a 0-a 5 by intraperitoneal injection (intraperitoneal injection), to thereby target the newborn cells (stimulating cells), and on day 23 of the experiment, the rats to be tested in groups a 0-a 5 were sacrificed, brain tissue sections having the hippocampal gyrus region (dentate gyrus) were selected and stained with BrdU, to thereby estimate the number of BrdU positive cells in the hippocampal gyrus.

(B) Intensity of epileptic seizure

The test evaluated the seizure intensity of rats in groups A0-A5 on days 1, 3, 5, 7, 9, 11, 13 and 21 of the test with a Racine score (Racine score) which was classified as 6: stage 1: mouth and face twitching (mouth and facial movement); stage 2: head nodding (head nodding); stage 3: myotonic seizures (myoclonic seizure); and 4, stage: forelimb amputation (forelimb donus) and hindlimb standing (stand on hindlimb); stage 5: muscle rigidity (muscular rigidity) and spasm (spasm); stage 6: and death.

Referring to fig. 3, the a0 th group of normal rats maintained at level 0 (as shown in "o" of fig. 3), the epileptic seizure intensity of the epileptic rats of groups a1 to a5 persisted at days 1, 3, 5, and 7 before the administration of clavulanic acid and/or valproic acid (as shown in "●" of fig. 3), while the epileptic seizure intensity of the epileptic rats of group a1 administered physiological saline and the epileptic rats of group a2 administered with low doses of clavulanic acid maintained at levels 3 to 4 (as shown in "" of fig. 3 and "", and the epileptic seizure intensity of the rats of group A3 administered with high doses of clavulanic acid slowed down (as shown in "3"), indicating that the administration of high doses of clavulanic acid can treat the epileptic seizure of the rats at days 9, 11, and 13; additionally, group A4 epileptic rats administered low doses of valproic acid were still ineffective in reducing the intensity of seizures due to insufficient administration of valproic acid (see "" ■ "") in FIG. 3. In addition, the seizure intensity of the epileptic rats of group A5 co-administered with low dose clavulanic acid and low dose valproic acid was significantly reduced (as shown in ""in FIG. 3), and the effect was significantly better than that of the low dose clavulanic acid alone (group A2) and the low dose valproic acid alone (group A4), indicating that both clavulanic acid and valproic acid had synergistic effects in treating seizures.

Furthermore, it is noted that, after 7 consecutive days of treatment (i.e., on days 7 to 13 of the trial), even if the treatment was stopped on days 14 to 20, the intensity of seizures (as shown in "x, and" ""in A3 group epileptic rats that had been administered a high dose of clavulanic acid, or in A5 group epileptic rats that had been administered a low dose of clavulanic acid together with a low dose of valproic acid) was significantly lower than the intensity of seizures (as shown in "" x "", and "" in FIG. 3) in A1 group epileptic rats that had been administered a physiological saline.

(C) Roller test

The motor function of the rats in groups A0-A5 was then tested by a roller test. In detail, in a training session (training session) of the first 3 days from the start of the experiment, rats not yet induced with PTZ (i.e., normal rats) were used as the rats R to be tested, and the rats R to be tested were walked on the roller W as shown in fig. 4a (the rotation speed of the roller was accelerated from 0rpm to 25rpm for 6 minutes each, and the training was performed for 3 times with a rest in between).

On the 14 th day of the experiment, rats in groups a 0-a 5 were used as the rats R to be tested, and the rats R to be tested were placed on the rollers W at a rotation speed of 25rpm for 3 minutes, respectively, and if the rats R to be tested lose their balance and fall off the rollers (as shown in fig. 4 b), it was revealed that the more serious the impairment of the coordination of movement (coordination) and balance ability (balance) of the rats R to be tested.

Referring to fig. 5, although the duration of exercise was significantly shorter in the epileptic rats (group a 1) administered with saline compared to the normal rats in group a0 (p < 0.01, compared to group a 0), the duration of exercise was significantly increased in the epileptic rats (group a2, no difference compared to group a 0; group A3, p < 0.05, compared to group a 1) administered with a low dose or a high dose of clavulanic acid. In addition, although the administration of low doses of valproic acid did not improve the impairment of motor coordination and balance ability in the epileptic rats (group a4, p < 0.001, compare to group a 0), the co-administration of low doses of clavulanic acid with low doses of valproic acid improved the impairment of motor coordination and balance ability in the epileptic rats (group a5, no difference compared to group a 0).

(D) Article identification test

On days 15-17 of the test, the damage status of the object recognition ability of rats R in groups A0-A5 was tested by the object recognition test. In detail, during the exposure period, the rat R to be tested is placed in an open box (open box) as shown in fig. 6a for 5 minutes, wherein three corners of the open box are respectively provided with objects (objects, hereinafter referred to as old objects O1, O2, O3) having the same size, color, shape and material and no peculiar smell.

On day 17 of the experiment, a test session was entered, the rats R to be tested were also placed in an open space box as shown in FIG. 6a, and the time (T) for the groups A0-A5 of rats R to be tested to explore the old object O1 was recorded_O1) And the total time (T) to explore the old objects O1, O2 and O3_O1+O2+O3) The percentage of time of exploration ((T) for the old part O1) for the rat R to be tested was calculated_O1/T_O1+O2+O3) X 100%); continuing for 5 minutes, replacing the old objects O1 with a new object O4 (shown in FIG. 6 b) having a different size, color, shape and material from the old objects O1, O2 and O3, and recording the new object O4 when the rats R to be tested are placed in the open space box, wherein the rats R to be tested in groups A0-A5 are searchedTime (T) of the new object O4_O4) And the total time (T) to explore the old objects O2, O3 and the new object O4_O2+O3+O4) To calculate the percentage of time of the new object O4 explored by the rat R to be tested ((T)_O4/T_O2+O3+O4)×100％)。

Referring to fig. 7, the percentage of time that the normal rats in group a0 were exploring the new device O4 was significantly greater than the old device O1, indicating that the normal rats could recognize the new device O4 in the environment (p < 0.001); and it was revealed that the recognition ability of the epileptic rats was impaired according to the test results of the epileptic rats (group a 1) administered with physiological saline, and thus the new material O4 could not be identified. Additionally, the epileptic rats (groups A2 and A3) administered low or high doses of clavulanic acid could recognize the new environmental substance O4 (group A2, p < 0.01; group A3, p < 0.05), and the administration of low doses of valproic acid failed to improve the injury status of the discriminatory ability of the epileptic rats (group A4). It is noted that co-administration of low doses of clavulanic acid and valproic acid improved the cognitive impairment in the epileptic rats (group a5, p 0.008) and was superior to low doses of clavulanic acid alone (group a 2) or low doses of valproic acid alone (group a 4), indicating that the combined administration of clavulanic acid and valproic acid had a synergistic effect on the treatment of cognitive impairment in epileptic subjects.

(E) Passive avoidance test

On days 18-20 of the test, passive avoidance tests were performed using a shuttle box (shuttle box) as shown in fig. 8 a-8 d to test the impairment of memory in rats of groups a 0-a 5.

The shuttle box includes a light chamber C1 (light chamber) and a dark chamber C2(dark chamber) separated by a sliding door d (guillotine door). First, in an exploration session (exploration session), as shown in fig. 8a, the sliding door D is opened, and the rat R to be tested is placed in the dark room C2, so that the rat R to be tested can freely shuttle and explore in the light room C1 and the dark room C2.

During the learning session (learning session), the sliding door D is closed, and the rat R to be tested is placed in the light chamber C1 as shown in fig. 8 b; then, after 30 seconds, the sliding door D is opened, and the rat R to be tested enters the dark room C2 immediately due to strong light avoidance (photophobism) as shown in fig. 8C. At this time, as shown in fig. 8D, the sliding door D is immediately closed, and an electric shock s (foot shock) is given to the rat R to be tested entering the dark chamber C2.

After a waiting period of 24 hours (latency), as also shown in fig. 8b, the rat R to be tested is placed in the bright room C1, the sliding door D is opened, and the latency (latency) of the rat R to be tested entering the dark room C2 is recorded, and if the latency is shorter, the memory impairment of the rat R to be tested is more serious.

Referring to FIG. 9, the saline-administered epileptic rats (group A1) had significantly shorter latency (p < 0.01, compared to group A0) than the normal rats of group A0, while the low dose of clavulanic acid administered epileptic rats (group A2) had shorter latency (group A2, p < 0.05, compared to group A0), and the administration of low dose of valproic acid failed to improve the memory impairment of the epileptic rats (group A4, p < 0.05, compared to group A0), however, the low doses of clavulanic acid and valproic acid administered together to the epileptic rats improved the memory impairment (p < 0.001 in group A5 compared to group A1; p < 0.01 compared to groups A2 and A4) in the epileptic rats, indicating that the combined administration of clavulanic acid and valproic acid had an additive effect in treating the memory impairment in the epileptic subjects.

(F) Histopathological analysis

On day 22 of the experiment, neonatal cells were labeled with 5-bromo-2 'deoxyuridine (BrdU) by intraperitoneal injection, and then on day 23 of the experiment, after sacrifice of the rats from group a0 to group a5 to be tested, the brains were subjected to coronal section (coronal section), and tissue sections with the region of the hippocampal dentate gyrus (dentate gyrus) were selected for 5-bromo-2' deoxyuridine (BrdU) staining, thereby estimating the number of neonatal BrdU positive cells in the hippocampal dentate gyrus, as shown in fig. 10.

Referring to fig. 10, the numbers of BrdU positive cells in the saline-administered epileptic rats (group a 1) were significantly decreased compared to the normal rats of group a0 (p < 0.05, compared to group a 0), indicating a loss of the newly-generated function of nerve cells; the administration of low doses of valproic acid (group A4) did not effectively increase neuronal neogenesis deficiency (p < 0.05, compared to group A0). However, both the low dose and high dose of clavulanic acid administered to the epileptic rats (group a2, group A3) and the co-administration of low dose clavulanic acid and low dose valproic acid (group a 5) restored the number of BrdU positive cells to normal values (no difference compared to group a 0).

Furthermore, it can be known that the dose to be administered is further converted from the formula of dose transfer (dose transfer) based on the Body Surface Area (BSA) (Reagan-Shaw et al (2007), FASEB J.,22: 659-661): when clavulanic acid and valproic acid are co-administered, the administration dosage of clavulanic acid is 0.016-10 mg/kg/day, and the administration dosage of valproic acid is 0.8-30 mg/kg/day; preferably, the administration dosage of the clavulanic acid is 0.016-4.99 mg/kg/day lower than the clinical dosage, and the administration dosage of the valproic acid is 0.8-19.99 mg/kg/day lower than the clinical dosage.

In summary, the compositions for treating epilepsy and the compositions for treating epilepsy-related dyskinesia, cognitive impairment and nerve injury of the invention can prevent epilepsy from occurring, weaken the intensity of epilepsy, and promote the regeneration of nerve cells in the dentate gyrus region of hippocampus through the synergistic effect of clavulanic acid and valproic acid, thereby treating epilepsy and treating epilepsy-related dyskinesia and cognitive impairment (e.g., impaired conditions that improve motor coordination and balance in epileptic individuals, impaired conditions that improve object recognition ability and memory ability in epileptic individuals).

In addition, the administration of the valproic acid and the clavulanic acid can reduce the administration dosage of the valproic acid, lead metabolic organs such as liver, kidney and the like to rapidly and efficiently metabolize and decompose the valproic acid, prevent the valproic acid from being excessively accumulated in organisms, reduce the nerve, spirit and behavior interference caused by the valproic acid, reduce the burden of the excessive administration of the valproic acid on the metabolic organs such as liver, kidney and the like and achieve the aim of reducing the possibility of side effects.

Meanwhile, compared with the existing antiepileptic drugs which are required to be continuously administered, the anti-epileptic drug can still maintain good effect of inhibiting the epileptic seizure even if the treatment is stopped under the condition that the clavulanic acid is independently administered or the clavulanic acid and the valproic acid are co-administered, and also has the effect of improving the epileptic-related dyskinesia and cognitive deficits.

It should be noted that the development of a therapeutic method for drugs is ultimately required to enter clinical trials to verify the efficacy of the drug in patients, and in clinical trials, the currently used drugs cannot be stopped based on the safety and interests of patients, so that the new drug to be discussed is usually combined with the currently used drugs (i.e., add-on study) in performing clinical trials, and the combined use effect of clavulanic acid and valproic acid is proved in the use of the composition of the present invention.