CN112794882B

CN112794882B - Polypeptide for treating epilepsy and pharmaceutical composition thereof

Info

Publication number: CN112794882B
Application number: CN202110193500.1A
Authority: CN
Inventors: 宋广福; 黄莉莉; 陈云; 于涛
Original assignee: Jiamusi University
Current assignee: Jiamusi University
Priority date: 2021-02-20
Filing date: 2021-02-20
Publication date: 2021-10-12
Anticipated expiration: 2041-02-20
Also published as: CN112794882A

Abstract

Disclosed is a composition for the treatment of epilepsy comprising carbamazepine and a specifically designed truncated variant conotoxin polypeptide; the polypeptide can obviously improve the anti-epileptic effect of carbamazepine. Also, the present invention provides a method for treating an epileptic disorder by administering the pharmaceutical composition to a patient in need thereof. The pharmaceutical composition has good epilepsy treatment effect and wide market prospect.

Description

Polypeptide for treating epilepsy and pharmaceutical composition thereof

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a polypeptide for treating epilepsy and application thereof.

Background

Epilepsy (epilesys), commonly known as epilepsy, is a chronic disease in which sudden abnormal discharges of cerebral neurons result in transient cerebral dysfunction. According to the latest Chinese epidemiological data, the total prevalence rate of domestic epilepsy is 7.0 per thousand, the annual incidence rate is 28.8/10 ten thousand, and the prevalence rate of active epilepsy with attacks within 1 year is 4.6 per thousand. Therefore, about 900 million epilepsy patients are estimated in China, 500-600 million of the epilepsy patients are active epilepsy patients, about 40 million epilepsy patients are newly added every year, and epilepsy has become the second most common disease of the neurology department, second to headache in China.

It is generally accepted that the incidence of epilepsy is age related. Mainly causes diseases in the juvenile age, the number of the diseases is reduced year by year along with the increase of the age, the number of the disease is 66 percent before 30 years old, and the number of the disease is less than 15 percent above 50 years old. The incidence of different seizure types of epilepsy is age-dependent. Researchers have found that the high incidence of childhood is mainly caused by birth injury, newborn asphyxia, congenital malformation and other reasons. The population of adults and middle-aged people is about to be stabilized at the level of 20-40/10 ten thousands of people. The types of seizures vary from age to age, as infantile spasms are seen only in infantile seizures. Typical absence seizures, while reflecting primary generalized epilepsy which may be persistent for life, are rare beyond childhood and adolescence.

Currently, there is no drug or method that is completely effective in treating epilepsy. Although there are already a number of drugs approved for the treatment of epilepsy, their pharmacological effects are often effective, with most drug response rates being less than 50%. And the side effects of the main antiepileptic drugs commonly used at present are more, and even the death of the patients can be caused when the side effects are serious.

Therefore, in the medical field, there is still a need for developing effective drugs or treatment methods for treating epilepsy, and the need is urgent. The present disclosure provides a polypeptide and a pharmaceutical composition thereof for treating epilepsy.

Disclosure of Invention

The invention provides a truncated variant conotoxin polypeptide from conus of general in south China sea, which can be used for treating epilepsy, and particularly can remarkably improve the curative effect of carbamazepine.

The existing research shows that some conotoxin mature peptides extracted from conus of south China sea can inhibit acetylcholine receptor. Wherein, the acetylcholine receptor can be a nerve type acetylcholine receptor or a muscle type acetylcholine receptor. And thus, researchers believe that the mature peptides can be used to treat neurological disorders such as neuropathic, addictive, epilepsy, and the like.

Conus belongs to Conidae (Conidae) of gastropoda, inhabits shallow sea water of tropical sea, and is named due to conical or conus shape. The Conotoxin is a main weapon for predation and defense, and is a cocktail-like mixed toxin consisting of a plurality of single toxic peptides, and the main component is active polypeptide compounds with high specificity to different ion channels and nerve receptors, namely Conotoxin (Conotoxin). The conotoxin is a small molecular polypeptide consisting of 10-30 amino acid residues. To date, there are nearly thousands of conotoxins that have been isolated, and tens of conotoxins have been patented in the united states patent. They have wide application values in analgesia, ischemic protection, epilepsy treatment, diagnosis of certain diseases and receptor research, and some have entered clinical research or have been officially approved by the FDA as new drugs for treatment, and are used as specific diagnostic reagents and analgesics. On the other hand, conotoxins have become standard tools for channel and receptor identification and diagnosis, such as voltage-gated calcium ion channels (VSCCs) and N-type acetylcholine receptors (nAChRs), as excellent probes or tools for studying ion channels and membrane receptors, and have been widely used in the field of neuropharmacology.

Conus is widely distributed in the south China sea and other sea areas, and more than 80 conus species are found. At present, the research on the biochemical structure and the neuropharmacological activity of the conotoxin is based on the molecular structure of naturally extracted toxin and is researched by using artificially synthesized toxin. According to previous researches, the conotoxin mature peptide has the activity of a nerve type acetylcholine receptor, and is generally considered to have a certain effect of treating epilepsy. When the inventor researches the related conotoxin mature peptide, the effect of the combination of the conotoxin mature peptide and carbamazepine on treating epilepsy is not greatly different from that of the carbamazepine alone. In the process of researching the conotoxin mature peptide, the inventor designs a section of truncated variant conotoxin polypeptide of the mature peptide, and the polypeptide is combined with carbamazepine for use, so that the anti-epileptic effect of the conotoxin can be remarkably improved, and the conotoxin mature peptide has good clinical application value. The sequence of the conotoxin mature peptide is DGCSNAGGFCGIHPGLCCSEICLVWCT, and the truncated variant conotoxin polypeptide NAGGKGGIHRGLCCSEICLV.

The invention also provides methods for treating epileptic disorders, including epilepsy (epilepsy), epilepsy with generalized tonic clonic seizures, epilepsy with myoclonic absence, frontal lobe epilepsy, temporal lobe epilepsy, and the like, by administering the pharmaceutical composition to a patient in need thereof.

Therefore, it is another object of the present invention to provide a pharmaceutical composition for treating epilepsy, comprising a truncated variant conotoxin polypeptide, preferably comprising and truncated variant conotoxin polypeptide and carbamazepine. Preferably, the weight ratio of the variant conotoxin polypeptide to carbamazepine in said medicament is 1:10-20, more preferably 1:12-16, most preferably 1: 15.

The amino acid sequence of the truncated variant conotoxin polypeptide of the invention is NAGGKGGIHRGLCCSEICLV.

Preferably, the pharmaceutical composition containing carbamazepine is prepared into a solid oral preparation. The composition can be prepared into solid oral dosage forms such as tablets, capsules and the like according to the weight ratio of the carbamazepine and the truncated variant conotoxin polypeptide.

Further preferably, the pharmaceutical composition comprising carbamazepine, wherein each unit of preparation comprises 100-200mg of carbamazepine and 5-20mg of truncated variant conotoxin polypeptide.

Still further preferably, the pharmaceutical composition comprising carbamazepine as described above, wherein said solid oral dosage form contains 100mg of carbamazepine per unit dosage form and 10-15mg of truncated variant conotoxin polypeptide.

Detailed Description

The present invention will be described in further detail with reference to examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example (b): comparative experiment of carbamazepine compound preparation

Healthy male mice 96, weighing 20-23g, were randomly divided into six groups after acclimatization for 3 days: normal control group, model control group, carbamazepine + conotoxin mature peptide, carbamazepine + truncated mutant conotoxin polypeptide (low dose), carbamazepine + truncated mutant conotoxin polypeptide (high dose), 16 in each group. The other five groups except the normal control group are used for establishing an epilepsy model by the following method: before the first medicine taking, pentaerythrine is injected into abdominal cavity according to body weight (50 mg/kg). Mice in each group were gavaged 1 time each in the morning, in the evening with the test substances and doses of Table 1 (administration volume 0.3-0.4m1) starting on day 2 after injection for 7 days. After the last administration for 4h, normal control group was injected with normal saline, and the other mice were injected with 50mg/kg of pentaerythrite in the 2 nd intraperitoneal injection.

Table 1: experimental groups and Experimental dosing regimens

Grouping	Sample size (only)	Dosage form
			Normal control group	16	0.4ml of pure water
Model control group	16	0.4ml of pure water
			Carbamazepine group	16	80 mg/kg-1/d-1 carbamazepine
Carbamazepine + mature peptide	16	80 mg/kg-1/d-1 carbamazepine +12mg mature peptide
			Carbamazepine + mutant peptide (Low)	16	80mg kg-1 d-1 carbamazepine +8mg mutant peptide
Carbamazepine + mutant peptide (high)	16	80mg kg-1 d-1 carbamazepine +12mg mutant peptide

After injection, all experimental mice were placed in the corresponding observation boxes, respectively, and the behavioral changes of the mice were observed. Seizure latency was recorded as the time from intraperitoneal injection to onset of epilepsy. The seizure latency of epilepsy was observed and recorded in groups of mice injected intraperitoneally with pentaerythrine 1 and 2 days after the 7d treatment. The seizure latency of the mice in each treatment group is obviously prolonged compared with the [ (78.3 +/-15.6) s ] of the model control group, wherein the effect of the carbamazepine + truncated mutant conotoxin polypeptide group is most obvious, the seizure latency of the mice in the group is (220.3 +/-19.8) s, and the seizure latency is significantly different from that of the carbamazepine group (P is less than 0.05). See table 2 for specific results.

Table 2: results of each group (latent period of epileptic seizure)

Grouping	Sample size (only)	Incubation period before administration	Incubation period after administration
				Model control group	16	76.1±15.2	78.1±15.8
Carbamazepine group	16	77.8±15.6	153.2±19.5
				Carbamazepine + mature peptide	16	77.1±16.3	158.1±21.2
Carbamazepine + mutant peptide (Low)	16	77.0±16.9	215.6±17.6
				Carbamazepine + mutant peptide (high)	16	76.8±17.7	220.3±19.8

After recording the latency data by intraperitoneal injection of pentylenetetrazol 2, the mice were immediately decapitated on ice to take brains, the hippocampus was separated, rinsed to drain water, weighed and stored in liquid nitrogen. According to the current theoretical study on epilepsy, in epileptic patients and animals, the brain hippocampus of the epileptic patients and animals has a significant increase in nitric oxide synthase NOS and NO, and the latter directly causes hippocampal cytotoxicity, leading to epilepsy. Thus, the isolated hippocampus was treated and examined for NOS activity and NO content therein. The detection result shows that the activity of NOS and the content of NO in hippocampal cells of a mouse with the carbamazepine + truncated mutant conotoxin polypeptide group are obviously reduced.

Table 3: content of NOS and NO in hippocampus of various groups of epileptic mice

Claims

1. A truncated variant conotoxin polypeptide for use in the adjuvant treatment of epilepsy, wherein the amino acid sequence of said polypeptide is represented as NAGGKGGIHRGLCCSEICLV.

2. A pharmaceutical composition for treating epilepsy, comprising carbamazepine and a truncated variant conotoxin polypeptide of claim 1.

3. The pharmaceutical composition of claim 2, wherein each unit of preparation comprises 100-200mg of carbamazepine and 5-20mg of the truncated variant conotoxin polypeptide.

4. A pharmaceutical composition according to claim 3, wherein each unit formulation contains 100mg of carbamazepine and 10-15mg of the truncated variant conotoxin polypeptide.

5. Use of a truncated variant conotoxin polypeptide of claim 1 and carbamazepine in the preparation of a medicament for the treatment of epilepsy.