Use of a pharmaceutical composition in the treatment of migraine

Abstract

The present invention relates to a pharmaceutical combination, and the pharmaceutical composition comprises cobra phospholipase A2 and its pharmaceutically acceptable carrier. This pharmaceutical composition can be used for preventing and / or treating migraine, and down-regulating the concentration level of CGRP in vivo, which has important clinical significance for the treatment and / or prevention of migraine.

Description

Technical Field

[0001] The present invention relates to the use of a composition in the preparation of a medicament for treating migraine in patients. Background Art

[0002] Migraine is a common and recurrent headache disorder, mainly characterized by severe throbbing headache on one or both sides, often accompanied by symptoms such as nausea, vomiting, and hypersensitivity to light and sound stimuli. Its common types include migraine without aura, migraine with aura, and chronic migraine, etc. The causes of migraine may be related to genetics, endocrine metabolism, environmental factors, mental factors, etc., and often have a genetic background. The number of female patients is 2-3 times that of male patients. Most cases start in adolescence, and a small number can occur in childhood. Symptomatically, the typical manifestation of migraine is severe headache, which can be pain in one side of the head and migrate to the other side, and may also be sensitive to light, noise, and odors. Common accompanying symptoms include nausea, vomiting, stomach discomfort, abdominal pain, feeling very hot or cold, and pale skin color.

[0003] In terms of treatment, currently migraine cannot be cured completely. Most treatment aims to relieve or terminate headache attacks, alleviate accompanying symptoms, and prevent headache recurrence. It includes drug treatment and non-drug treatment. Among them, drug treatment is divided into treatment during the attack period and preventive treatment. Migraine has always been a clinical symptom that seriously affects the quality of human life. Although there are drugs for treating migraine clinically, such as non-steroidal analgesics, monoclonal antibodies, etc., they cannot produce satisfactory treatment effects, and some drugs have very serious side effects. Frequent use will cause serious clinical toxic and side effects to patients. Therefore, the current drugs for treating migraine have not really reached the level of meeting the clinical needs of patients.

[0004] Further research on the causes of migraine has found that the causes of migraine are related to some pathological factors such as the increase in the concentration of calcitonin gene-related peptide (CGRP). Treatment methods such as reducing the level of calcitonin gene-related peptide (CGRP) or antagonizing the CGRP receptor are effective in improving the pain degree of migraine (1-5). Currently, the treatment regimens for migraine mainly include acute and preventive treatments. With the exploration of signal molecules related to migraine attacks, more and more treatment methods have been developed for migraine treatment. Downregulating the signal pathway of the calcitonin gene-related peptide (CGRP) receptor has attracted much attention as a new migraine treatment method. Summary of the Invention

[0005] The present invention relates to the application of a composition in the preparation of a medicament for treating migraine in patients, characterized in that the composition contains cobra phospholipase A2, and the pharmaceutical composition of the present invention has an analgesic effect on a rat nitroglycerin-induced migraine test model.

[0006] Phospholipase A2 (PLA2) is a widely distributed enzyme family that exists in various animal tissues, especially in the venoms of animals such as snake venom (type I) and the pancreatic secretions of mammals (type II). The main structural feature of cobra snake phospholipase A2 is a platform of two anti-parallel long-chain helical structures connected by disulfide bonds. Their amino acid sequences maintain a high degree of homology and some functional structural similarities. They contain 7 disulfide bonds in their molecules, and they all have the typical 11th - 77th characteristic disulfide bond of type I PLA2. Their mature proteins have a high degree of homology in the amino acid sequence, with a molecular weight of about 12 - 14 KD, having 118 or 119 amino acid residues. The common functional structure enables them to have common pharmacological activities, such as reducing the concentration of CGRP in the body. Inhibiting the concentration of CGRP will improve the pain level of migraine. Therefore, the same functional structure of cobra snake phospholipase A2 results in similar pharmacological functions.

[0007] Our animal model study first found that cobra snake phospholipase A2 has an analgesic effect on the rat nitroglycerin-induced migraine test model. Its mechanism of action may be related to blocking or downregulating the signal pathway of calcitonin gene-related peptide (CGRP). Cobra snake phospholipase A2 can treat and prevent the occurrence of migraine at the same time, and has no serious toxic side effects in animal experiments. Therefore, it is a promising drug candidate.

[0008] The amino acid sequence (FASTA) of the cobra snake phospholipase A2 mature protein with the effect of improving the pain level of migraine has the following common characteristics: (SEQ ID No.1) nly qfknmiqctv psrswwdfad ygcycgkggs gtpvddldrccqvhdxcyxe aekisgcwpy xktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pyxxxxynixlkarcq

[0009] Furthermore, in some embodiments, phospholipase A2 with the following 4 amino acid sequences all showed a common analgesic effect on the rat nitroglycerin-induced migraine test model.

[0010] (SEQ ID No.2)

[0011] nly qfknmiqctv psrswwdfad ygcycgrggs gtpvddldrc cqvhdncyne aekisgcwpyfktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pynnnnynid lkarcq

[0012] (SEQ ID No.3)

[0013] nly qfknmiqctv psrswwdfad ygcycgrggs gtpvddldrc cqvhdhcyne aekisgcwpysktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pynnnnynid lkarcq

[0014] (SEQ ID No.4)

[0015] nly qfknmvqctv pnrswwdfad ygcycgrggs gtpvddldrc cqvhdncyge aekisrcwpyfktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pyndnnynid lkarcq

[0016] (SEQ ID No.5)

[0017] nlyqfknmiq ctvpsrswwd fadygcycgr ggsgtpvddl drccqvhdnc yneaekisgcwpyfktysye csqgtltckg gnnacaaavc dcdrlaaicf agapyndndy ninlkarc

[0018] Cobra phospholipase A2 can be extracted from snake venom or obtained by recombinant or synthetic techniques.

[0019] In some embodiments, the amino acid sequence of cobra phospholipase A2 in the composition is SEQ ID No2.

[0020] In some embodiments, the amino acid sequence of cobra phospholipase A2 in the composition is SEQ ID No3.

[0021] In some embodiments, the amino acid sequence of cobra phospholipase A2 in the composition is SEQ ID No4.

[0022] In some embodiments, the amino acid sequence of cobra phospholipase A2 in the composition is SEQ ID No5.

[0023] In some embodiments, the composition is administered once daily for 3 - 120 consecutive days.

[0024] In some embodiments, the composition is administered once every 12 hours, twice a day, for 3 to 120 consecutive days.

[0025] In some embodiments, the composition is administered once every 4 hours, three times a day, for 3 to 120 consecutive days.

[0026] In some embodiments, the dosage of cobra phospholipase A2 in the composition is 5.0 - 100 micrograms per kilogram per administration.

[0027] In some embodiments, the dosage of cobra phospholipase A2 in the composition is 80 micrograms per kilogram per administration.

[0028] In some embodiments, the dosage of cobra phospholipase A2 in the composition is 60 micrograms per kilogram per administration.

[0029] In some embodiments, the dosage of cobra phospholipase A2 in the composition is 40 micrograms per kilogram per administration.

[0030] In some embodiments, the dosage of cobra phospholipase A2 in the composition is 20 micrograms per kilogram per administration.

[0031] In some embodiments, the dosage of cobra phospholipase A2 in the composition is 10 micrograms per kilogram per administration.

[0032] In some embodiments, the dosage of cobra phospholipase A2 in the composition is 5.0 micrograms per kilogram per administration.

[0033] In some embodiments, the dosage form of the composition is a sublingual film.

[0034] In some embodiments, the dosage form of the composition is an oral dosage form.

[0035] In some embodiments, the dosage form of the composition is an injection dosage form.

[0036] In some embodiments, the dosage form of the composition is a nasal spray dosage form.

[0037] In some embodiments, the dosage form of the composition is a transdermal dosage form.

[0038] In some embodiments, the pharmaceutically acceptable excipients are selected from one or more of protein stabilizers, film formers, mucosal penetration enhancers, solubilizers, solvents, or preservatives.

[0039] In some embodiments, the protein stabilizer is mannitol.

[0040] In some embodiments, the film excipients are propylene glycol, polyethylene glycol, hydroxypropyl-β-cyclodextrin, Tween 80, hydroxypropyl methylcellulose (HPMC), methylcellulose, xanthan gum.

[0041] In some embodiments, the mucosal penetration enhancers are azone, poloxamer, borneol, and dextrorboneol.

[0042] In some embodiments, the cosolvent is propylene glycol.

[0043] In some embodiments, the solvents are sodium chloride and ammonium acetate.

[0044] In some embodiments, the preservatives are benzyl alcohol and benzalkonium chloride.

[0045] Another advantage of the present invention lies in production. Since the cobra phospholipase A2 disclosed in the present invention has a clear amino acid sequence, it can be produced through genetic engineering, thus solving the practical problem of scarce snake venom resources. Even if phospholipase A2 is still obtained by separating and purifying natural snake venom, due to the clear amino acid sequence in the process, it is easier to control the quality and purity, which lays a necessary foundation for the development of drugs from monomer components in snake venom.

[0046] The following further illustrates the present invention with specific embodiments, but the following embodiments do not limit the present invention; at the same time, all equivalent substitutions in the art in accordance with the disclosed content of the present invention fall within the protection scope of the present invention. Detailed Description of the Invention

[0047] Examples:

[0048] Example 1: Obtaining of cobra phospholipase A2 protein (SEQ ID No.2)

[0049] Dissolve 1 g of cobra crude venom in 25 ml of 0.025 mol / L ammonium acetate buffer solution with pH 6.0, centrifuge at low temperature, and take the supernatant; equilibrate the TSK CM-650(M) column with 0.025 mol / L ammonium acetate solution with pH 6.0; after loading the sample, perform two-step gradient elution with 0.1 - 0.5 mol / L and 0.7 - 1.0 mol / L ammonium acetate buffer solution with pH 5.9 (0.1 - 0.5 mol / L and 0.6 - 1 mol / L), ultraviolet detection parameters: 280 nm; elution flow rate: 48 ml / h; collect various toxin components according to the recorded chromatogram, 12 protein peaks are eluted in the collected solution, perform N-terminal sequencing on each protein peak, and further sequence the protein with N-terminal nlyqfknm, and finally obtain various sequence protein solutions of cobra phospholipase A2.

[0050] The amino acid sequence of the primary structure of cobra phospholipase A2 (SEQ ID No.2) obtained by sequencing in Fasta format is as follows:

[0051] nly qfknmiqctv psrswwdfad ygcycgrggs gtpvddldrc cqvhdncyne aekisgcwpyfktysyecsq gtltckggnn acaaavcdcd rlaaicfaga pynnnnynid lkarcq

[0052] Example 2: Analgesic effect of cobra phospholipase A2 on rats with nitroglycerin-induced experimental migraine model

[0053] 1. Experimental animals and modeling groups

[0054] Forty 180 - 220 g SD rats were randomly divided into 10 rats in the normal control group, 10 rats in the model control group, 10 rats in the PLA2 treatment group, and 10 rats in the PLA2 prevention group.

[0055] Modeling method: Nitroglycerin was intraperitoneally injected at a dose of 10 mg / kg for 3 consecutive days.

[0056] In the PLA2 prevention group, nasal drops were administered continuously for 7 days before modeling until the day of modeling, 100 micrograms / kg each time, 2 times a day. When modeling, except for normal saline in the normal group, in the model control group, PLA2 prevention group, and PLA2 treatment group, nitroglycerin was intraperitoneally injected at a dose of 10 mg / kg for 3 consecutive days as described above. In the PLA2 treatment group, the drug was administered simultaneously during modeling, 100 micrograms / kg each time, 2 times a day. Cobra phospholipase A2 (PLA2) SEQ ID No.2 was used for animal experiments.

[0057] 2. Observation indicators and detection methods

[0058] 2.1 The number of head scratching of rats at each time period was used as an observation indicator. The ethological indicators of animals were observed 30 - 120 minutes after the last modeling. Starting from the last modeling, every 30 minutes was taken as a time period, and the head scratching results of rats after modeling were observed using the method of continuous duration segmented counting. (Shown in Table 1)

[0059] 2.2 The serum CGRP concentration level was used as an observation indicator. After the ethological detection of rats, each group of mice was anesthetized, blood was taken from the periorbital area, allowed to stand, centrifuged at 3000 r / min for 20 minutes at 4°C, the supernatant was obtained, stored in a -80°C refrigerator, and detected according to the instructions of the ELISA kit.

[0060] 3. Experimental results

[0061] Table 1 Number of head scratching of rats

[0062] ( n = 10)

[0063] Grouping 30 (min) 60 (min) 90 (min) 120 (min) Normal group 0.93 ±0.43 0.77 ±0.34 1.16 ±0.65 0.58 ±0.34 Model group control 31.4 ±4.63 42.3 ±3.60 39.8 ±3.48 22.5 ±3.58 PLA2 prevention group 27.4 ±3.04* 38.3 ±4.10* 35.6 ±4.588 19.2 ±2.74* PLA2 treatment group 32.3 ±3.34 39.2 ±2.1* 32.3 ±5.90* 16.2 ±3.95**

[0064] * indicates significant differences between the PLA2 treatment group and the prevention group compared with the model control group (P < 0.05.); ** indicates significant differences between the PLA2 treatment group and the model control group (P < 0.01).

[0065] Table 2 Comparison of blood CGRP concentration levels in rats of each group

[0066] (pg / m1 n = 10)

[0067] Grouping CGRP pg / ml Normal group 220.7 ±25.67 Model group control 321.5 ±41.11 PLA2 prevention group 282.5 ±34.67* PLA2 treatment group 270.6 ±36.37*

[0068] * indicates significant differences between the PLA2 treatment group and the PLA2 prevention group compared with the model control group (P < 0.05.)

[0069] Reference:

[0070] 1. Anne - Sophie Wattiez et al;. Calcitonin gene - related peptide (CGRP): Role in migraine pathophysiology and therapeutic targeting. Expert Opin Ther Targets. 2020 Feb; 24(2): 91–100.

[0071] 2. Andrew Charles et al;. Targeting calcitonin gene - related peptide: a new era in migraine therapy. Lancet. 2019 Nov 9; 394(10210): 1765 - 1774.

[0072] 3. Li Xue et al, Research progress on the role of calcitonin gene - related peptide in migraine and its targeted drugs. Progress in Pharmaceutical Sciences. 2024, 48(02)

[0073] 4. Zeng Junsheng et al, Study on the effects of combined use of Ginkgo biloba capsules and flunarizine hydrochloride on intracranial arterial hemodynamics and serum β - EP, 5 - HT, CGRP levels in migraine patients. Acta Neuropharmacologica. 2023(03)

[0074] 5. Chen Gangxing et al., Effects of Modified Pinellia and Atractylodes Rhizome Decoction Combined with Aspirin Enteric-coated Tablets on Headache Symptoms and Plasma 5-HT, VEGF, and CGRP in Migraine Patients. Sichuan Journal of Traditional Chinese Medicine. 2024(06).

Claims

1. Use of a composition in the preparation of a medicament for treating and / or preventing migraine, said composition containing a therapeutically effective dose of cobra phospholipase A2 and pharmaceutically acceptable excipients; the amino acid sequence of the mature protein of said cobra phospholipase A2 is as shown in SEQ ID No.

2.

2. The application according to claim 1, wherein Said medicament has at least one of the following applications (1)-(3): (1) Said medicament is used for treating migraine; (2) Said medicament is used for preventing migraine; (3) Said medicament is used for down-regulating the concentration of calcitonin gene-related peptide in the body of a migraine patient.

3. The application according to claim 1, characterized in that, The dosage form of said composition is an oral dosage form, a sublingual dosage form, a nasal dosage form, a transdermal dosage form or an injection dosage form.

4. The application according to claim 1, characterized in that, Said pharmaceutically acceptable excipients are selected from one or more of protein stabilizers, film formers, mucosal permeation enhancers, solubilizers, solvents, preservatives.

5. The application according to claim 1, further characterized in that the protein stabilizer among said pharmaceutically acceptable excipients is mannitol, the film former is propylene glycol, polyethylene glycol, hydroxypropyl-β-cyclodextrin, Tween-80, hydroxypropyl methylcellulose, methylcellulose, xanthan gum, the mucosal permeation enhancer is laurocapram, poloxamer, borneol, isoborneol, the solubilizer is propylene glycol, the solvent is sodium chloride, ammonium acetate, and the preservatives are benzyl alcohol and benzalkonium chloride.

6. The application according to claim 1, characterized in that, The dose of cobra phospholipase A2 in said composition ranges from 5.0 μg / kg to 100 μg / kg per administration, and is administered 1-3 times a day.