CN110893183A

CN110893183A - Application of ginkgolide in preparation of medicine for preventing, relieving or treating amyotrophic lateral sclerosis

Info

Publication number: CN110893183A
Application number: CN201811066459.6A
Authority: CN
Inventors: 刘科; 邬春久; 李慧琴; 鄢云彪
Original assignee: Chengdu Baiyu Pharmaceutical Co Ltd
Current assignee: Chengdu Baiyu Pharmaceutical Co Ltd
Priority date: 2018-09-13
Filing date: 2018-09-13
Publication date: 2020-03-20

Abstract

The invention discloses an application of ginkgolide in preparing a medicine for preventing, relieving or treating amyotrophic lateral sclerosis, belonging to the field of medicines. The ginkgolide is selected from one or more of bilobalide or ginkgolide B or ginkgolide A or ginkgolide C or ginkgolide M or ginkgolide J or ginkgolide K or ginkgolide L or ginkgolide N or ginkgolide P or ginkgolide Q. The invention discloses a ginkgolide composition and a ginkgolide monomer which can improve the movement function of ALS transgenic mice to different degrees and have good improvement effect on female mice and male mice; the invention also discloses a ginkgolide composition, ginkgolide B and bilobalide which can delay the morbidity time of the ALS transgenic mice and have good delay effect on female mice and male mice, so that the ginkgolide composition can be further used for preventing, relieving or treating the amyotrophic lateral sclerosis of mammals. The invention provides a new and safe drug choice for treating amyotrophic lateral sclerosis.

Description

Application of ginkgolide in preparation of medicine for preventing, relieving or treating amyotrophic lateral sclerosis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of ginkgolide in preparation of a medicine for preventing, relieving or treating amyotrophic lateral sclerosis.

Background

Amyotrophic Lateral Sclerosis (ALS) is also known as Luga Rayleigh Disease (Lou Gehrig's Disease) or Charcot-tooth Disease (Maladiede Charcot). The primary disease marker is progressive degeneration of the superior and inferior motor neurons in the corticospinal tract. Dysfunction of the lower motor neurons (in the brainstem and spinal cord) triggers general debilitation, muscular atrophy and paralysis. Failure of the respiratory muscles is generally a fatal event, occurring within 1-5 years of onset. ALS is the most common motor neuron disease in adults, affecting approximately 30,000 in the united states and 5,000 in the uk each year (Leigh and Swash, 1991). Typical onset ages are between 50 and 70 years of age, although sometimes occur at younger ages. Most cases (90-95%) are classified as sporadic als (sals), with the remainder inherited, called familial als (fals). However, the exact cause of most cases is still unknown and there is no effective treatment to stop the progression of the disease.

In patent CN101675932B, the inventor uses CD11b and GFAP as markers to observe the forms of microglia and astrocyte at the anterior spinal cord horn of SOD1-G93A transgenic mice respectively, and finds that the glia cells including microglia and astrocyte at the anterior spinal cord horn of SOD1-G93A transgenic mice are widely activated at 120 days, and the transgenic SOD1-G93A mice have iNOS and TNF- α levels which are obviously increased compared with wild mice, and patent CN106138076A discloses that ginkgo biloba extract flavonoid glycoside dimers biginkgosides D and H have neuroanti-inflammatory activity, can obviously inhibit the release of NO in the microglia (BV2) activated by Lipopolysaccharide (LPS), and can be further used for preparing medicines for treating neurodegenerative diseases, such as ALS.

The main active ingredients in the ginkgo leaf are flavonoids and ginkgo terpene lactones, and recent researches show that the ginkgo leaf extract has a treatment effect on ALS mainly through the flavonoids which are important effective parts of the ginkgo leaf extract. In addition to the above mentioned ginkgo biloba flavonoid glycoside dimers D and H as anti-inflammatory actives for the treatment of ALS, patent CN102159203A also discloses the flavonoid ginkgetin K as an HDAC1 activator for the treatment of ALS. The other effective part of ginkgo terpene lactones of ginkgo biloba leaf extract is disclosed in the literature to be used for preventing and treating ALS. Related studies show that EGb761 has obvious protective effect on ALS transgenic mice, but the effect has obvious sex difference. Specifically, EGb761 has a significant improvement in locomotor ability and survival rate in male G93A transgenic ALS mice, and can combat loss of anterior spinal cord neurons, but only improve survival rate in littermate female G93A transgenic ALS mice without significant difference. None of the prior studies disclose a specific effective fraction of ginkgo biloba extract to exert therapeutic effects (Rober j. ferrante et al, 2001).

Meanwhile, as disclosed in patent CN101675932B, ALS has different pathogenesis compared with other neurodegenerative diseases such as parkinson's disease or alzheimer's disease, and lower motor neurons are more susceptible and progress rapidly, so that the therapeutic effect of drugs for treating neurodegenerative diseases such as alzheimer's disease on ALS cannot be predicted. Patent CN105832944B discloses that animal experiments and clinical studies only prove that a few chemical drugs, such as riluzole, edaravone, sodium valproate, etc., have certain curative effects on amyotrophic lateral sclerosis. The first of these drugs, Riluzole (Riluzole) approved by the FDA for marketing, only increases the annual chance of survival by about 10%.

Therefore, research and development of drugs suitable for ALS are urgent problems to be solved, and more drug choices are provided for ALS patients.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of ginkgolide in preparing a medicament for preventing, relieving or treating amyotrophic lateral sclerosis of mammals.

In one aspect, the present invention provides the use of ginkgo terpene lactones in the manufacture of a medicament for the prevention, alleviation or treatment of amyotrophic lateral sclerosis in a mammal.

The term "prevention" as used herein refers to the prevention of disease occurrence and/or the prevention of disease recurrence and/or the prevention of disease-related symptom occurrence and/or recurrence.

The term "treating" as used herein refers to reversing, alleviating or inhibiting the progression of one or more symptoms of the disease for which the term is applied.

The prevention, alleviation or treatment according to the present invention may be performed in an acute or chronic manner. Depending on the condition of the subject, preventive and/or palliative and/or curative measures are taken.

Further, the sex of the mammal according to the present invention is female or male.

Further, the ginkgolide is one of bilobalide or ginkgolide A or ginkgolide B or ginkgolide C or ginkgolide M or ginkgolide J or ginkgolide K or ginkgolide L or ginkgolide N or ginkgolide P or ginkgolide Q with an optional effective amount.

Preferably, the ginkgolide refers to an effective amount of bilobalide.

Preferably, the ginkgolide refers to an effective amount of ginkgolide B.

Further, the ginkgolide is selected from two or more of bilobalide or ginkgolide A or ginkgolide B or ginkgolide C or ginkgolide M or ginkgolide J or ginkgolide K or ginkgolide L or ginkgolide N or ginkgolide P or ginkgolide Q with effective dose.

Preferably, the ginkgolide is optionally an effective amount of two or more of bilobalide or ginkgolide a or ginkgolide B or ginkgolide C or ginkgolide J.

More preferably, the ginkgolide comprises an effective amount of bilobalide and ginkgolide a.

More preferably, the ginkgolide comprises an effective amount of bilobalide and ginkgolide C.

More preferably, the ginkgolide comprises an effective amount of ginkgolide B and ginkgolide A.

More preferably, the ginkgolide comprises an effective amount of ginkgolide B and ginkgolide C.

Preferably, the ginkgolide comprises an effective amount of bilobalide and ginkgolide B.

Preferably, the ginkgolide comprises an effective amount of bilobalide, ginkgolide B and ginkgolide a.

Preferably, the ginkgolide comprises an effective amount of bilobalide, ginkgolide B and ginkgolide C.

Preferably, the ginkgolide comprises an effective amount of bilobalide, ginkgolide a, ginkgolide B and ginkgolide C.

Preferably, the ginkgolide comprises an effective amount of bilobalide, ginkgolide a, ginkgolide B, ginkgolide C and ginkgolide J.

The term "effective amount" as used herein refers to an effective amount of a drug, which is administered in an amount that is responsive to the human and/or animal body, is prophylactic or therapeutic for the disease, and is acceptable to humans and/or animals. Further, the "effective dose of the drug" is administered in accordance with good medical practice, and takes into consideration the clinical condition of the subject, the site and method of administration, the dose, the age, sex, body weight of the patient, and other factors known to the physician.

Furthermore, the medicine is a pharmaceutical composition prepared from ginkgolide and/or pharmaceutically acceptable salts or hydrates thereof and/or isotopically labeled ginkgolide and pharmaceutical excipients or auxiliary components according to a conventional method.

In one embodiment of the present invention, the isotopically labeled ginkgolide is one or more of the ginkgolide monomers contained in the composition which is isotopically labeled.

The pharmaceutical composition is tablet, capsule, granule, pill, injection, and powder for injection.

Further, the amyotrophic lateral sclerosis is familial amyotrophic lateral sclerosis.

In an embodiment of the present invention, the active ingredient of the pharmaceutical composition further includes a pharmaceutically acceptable salt or hydrate of the above compound.

The pharmaceutically acceptable salt may be selected from the hydrochloride, hydrobromide, hydrofluoride, sulfate, nitrate, phosphate, formate, acetate, propionate, oxalate, malonate, succinate, fumarate, maleate, lactate, malate, tartrate, citrate, picrate, methanesulfonate, ethanesulfonate, benzenesulfonate, aspartate or glutamate of said compound.

In one embodiment of the present invention, the active ingredient compound of the above-mentioned pharmaceutical composition, or a pharmaceutically acceptable salt thereof, is further isotopically labeled, which means the same as those listed herein but wherein one or more atoms are replaced by another atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Isotopes that can be incorporated into compounds include hydrogen, carbon, nitrogen, oxygen, sulfur, i.e., 2H, 3H, 13C, 14C, 15N, 17O, 18O, 35S. Compounds containing the aforementioned isotopes and/or other atomic isotopes, and stereoisomers thereof, as well as pharmaceutically acceptable salts of such compounds, stereoisomers, are intended to be included within the scope of the present invention.

In a specific embodiment of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants or auxiliary components.

The pharmaceutically acceptable auxiliary materials refer to substances contained in the dosage form except for the active ingredients, and include, but are not limited to, fillers (diluents), lubricants (glidants or anti-adherents), dispersing agents, wetting agents, binders, regulators, solubilizers, antioxidants, bacteriostats, emulsifiers, disintegrants and the like. The binder comprises syrup, acacia, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethylcellulose, ethyl cellulose or hydroxypropyl methylcellulose), gelatin slurry, syrup, starch slurry or polyvinylpyrrolidone; the filler comprises lactose, sugar powder, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salt (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; the lubricant comprises superfine silica gel powder, magnesium stearate, talcum powder, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol and the like; the disintegrating agent comprises starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinylpyrrolidone or microcrystalline cellulose, etc.; the wetting agent comprises sodium lauryl sulfate, water or alcohol, etc.; the antioxidant comprises sodium sulfite, sodium bisulfite, sodium pyrosulfite, dibutylbenzoic acid, etc.; the bacteriostatic agent comprises 0.5% of phenol, 0.3% of cresol, 0.5% of chlorobutanol and the like; the regulator comprises hydrochloric acid, citric acid, potassium (sodium) hydroxide, sodium citrate, and buffer (including sodium dihydrogen phosphate and disodium hydrogen phosphate); the emulsifier comprises polysorbate-80, sorbitan fatty acid, pluronic F-68, lecithin, soybean lecithin, etc.; the solubilizer comprises Tween-80, bile, glycerol, etc.

The pharmaceutically acceptable auxiliary components have certain physiological activity, but the addition of the components does not change the dominant position of the pharmaceutical composition in the process of treating diseases or improving the physiological function of a human body, and only play auxiliary effects, and the auxiliary effects are only the utilization of the known activity of the components and are the conventional auxiliary modes in the field of medicine or health care. It is within the scope of the present invention to combine the aforementioned auxiliary components with the compounds or compositions of the present invention.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses a ginkgolide composition and a ginkgolide monomer which can improve the movement function of ALS transgenic mice to different degrees and have good improvement effect on female mice and male mice.

The invention discloses a ginkgolide composition, ginkgolide B and bilobalide which can delay the morbidity time of ALS transgenic mice and have good delay effect on female mice and male mice.

Meanwhile, the experimental animals have no obvious abnormality before and after the experiment, and the ginkgolide has good safety when being used for treating ALS.

The research result of the invention widens the application range of the ginkgolide and the composition thereof, and provides a new medicine selection for treating amyotrophic lateral sclerosis.

Detailed Description

The inventor of the invention discovers for the first time that ginkgolide has a positive effect on improving the motor function of the ALS transgenic mice and has no sex difference through intensive research, further researches discover that ginkgolide also has the beneficial effects of delaying the onset of disease and prolonging the survival time of the ALS transgenic mice, and further researches discover that ginkgolide composition, ginkgolide B and bilobalide have no sex difference on delaying the onset of disease of the ALS transgenic mice.

The ginkgolide can be a ginkgolide monomer or a composition consisting of ginkgolide monomers. The ginkgolide monomer can be obtained by purchasing a commercially available product or prepared by separating and purifying by the existing method. Specifically, the ginkgolide monomer can be ginkgolide A, B, C, M, J, K, L, N, P, Q or ginkgolide monomer compounds such as bilobalide and the like. Wherein ginkgolide A, B, C, M, J, K, L, N, P, Q can also be collectively called ginkgolide.

The structure of the ginkgolide is composed of 6 oxygen-rich five-membered rings (including a spiro [4.4] -nonane ring, a tetrahydrofuran ring and three lactone rings), 10-12 stereocenters and a special tert-butyl group. The isolated ginkgolides differ only in the number and position of hydroxyl groups in structure. The bilobalide named by the invention contains three lactone rings and a tertiary butyl group, only has one all-carbon ring, and belongs to sesquiterpene lactone.

When the ginkgolide of the present invention comprises two or more ginkgolide monomer compounds, it can be prepared by combining the corresponding monomer compounds.

The terms "subject", "individual" or "patient" are used interchangeably herein and include mammals, including primates.

The term "amyotrophic lateral sclerosis" described in the present invention is equivalent to "ALS", to "luga lei disease", to "charcot disease", and to "progressive freezing disease". ALS is characterized by motor neuron death in the cortex, brainstem and spinal cord. In the case of ALS, about 5-10% are familial (fALS) with the remainder sporadic (sALS). In a subset of ALS familial patients, mutations were found in the gene encoding copper-zinc superoxide dismutase 1(SOD 1). SOD1 is a cytosolic enzyme involved in free radical detoxification. In addition, overproduction of the allele encoding the pathogenic human SOD1 protein in motor neurons leads to late-onset progressive neurodegenerative diseases. Examples of alleles encoding pathogenic SOD1 proteins include, but are not limited to, SOD1G93A, SOD1G85R, and SOD1G 37R.

The invention selects SOD1G93A transgenic mice as ALS disease animal models to research and demonstrate the prevention, alleviation or treatment effect of ginkgolide on ALS.

In the research process, the inventor discovers that ginkgolide monomers bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, ginkgolide K, ginkgolide L, ginkgolide M, ginkgolide N, ginkgolide P and ginkgolide Q have different degrees of improvement on the movement symptoms, the morbidity time and the survival time of ALS transgenic mice, wherein the bilobalide and ginkgolide B have the optimal effects, the ginkgolide A and ginkgolide C have the inferior effects, and other ginkgolide monomers have certain effects. Therefore, in the experimental example of the invention, bilobalide B, bilobalide A, bilobalide C and bilobalide J are selected as the bilobalide monomer group to further explain the improvement effect of the bilobalide monomer on the survival quality and the survival period of the ALS transgenic mice. Selection of test examples of the invention

The ginkgolide injection is used for researching the improvement effect of the ginkgolide composition on the survival quality and the survival period of ALS transgenic mice. Developed by Chengdu Baiyu pharmaceutical products Limited company

The ginkgolide injection comprises the following effective components: 48% of bilobalide, 51% of bilobalide (GA, GB, GC), and the total amount of effective components is more than 99%.

The inventors have surprisingly found that, unlike prior art reports, there is no significant sex difference in the improvement of locomotor function of ALS transgenic mice by the ginkgolide composition and its monomers, and that there is a delay in the onset time of ALS transgenic mice by the ginkgolide composition, ginkgolide B, bilobalide and there is no significant sex difference in this effect.

The advantageous effects of the present invention are further described below by way of test examples.

Test example 1 Effect of ginkgolides on the improvement of the quality and Life cycle of ALS transgenic mice

1. Materials and methods

1.1 Experimental animals: SOD1G93A mice, male and female halves, purchased from Nanjing biomedical research institute, animal quality certification number (SCXK (Su) 2015-0001), were routinely raised in SPF-level laboratory animal houses. Animals were fed ad libitum for more than 1 week prior to the experiment.

1.2 reagents and drugs: riluzole (seirofei pharmaceutical co., ltd.), bilobalide injection and monomers bilobalide a, bilobalide B, bilobalide C, bilobalide J, bilobalide were provided by the gumbo pharmaceutical co., ltd., experimental setup 8 groups: model control group (saline), positive control group (riluzole, 26mg/kg, gavage), bilobalide group (bilobalide injection, 14mg/kg), bilobalide group a (bilobalide a, 14mg/kg), bilobalide group B (bilobalide B, 14mg/kg), bilobalide group C (bilobalide C, 14mg/kg), bilobalide group J (bilobalide J, 14mg/kg), bilobalide group (bilobalide, 14mg/kg), intraperitoneal injection, once a day. The drugs were all formulated to the corresponding concentrations with normal saline.

1.3 method: animals were administered in groups before onset (79 days of age), and were randomized into 8 groups of 12 animals each, half male and female, based on body weight.

1.4 symptom score and time to onset: and 4, dividing: when the mouse is suspended, the hind legs are extended and stay for 2-3 times for no less than 2 seconds; indicating normal. And 3, dividing: hind limb adduction (weakness) or tremor occurs when the mouse is suspended; indicating onset of disease. And 2, dividing: walking 10cm of toe and curling at least twice or dragging the foot; the water is put to the place where the animals are easy to eat. 1 minute: the two hind limbs are completely paralyzed and can crawl only by the forelimbs. 0 minute: the two hind limbs are completely paralyzed, and the mouse can not turn over within 30 seconds after lying on the back; death is defined. The time to onset of disease was recorded for the test animals.

1.5 survival observations: the animal mortality time was recorded and the mean survival time of the test animals was calculated.

2. Results of the experiment

Results of the experiment to

The clinical symptom score is judged by adopting rank sum test, other groups adopt single-factor variance analysis to judge the statistical significance of mean difference, and P is less than 0.05, so that the statistical difference exists.

TABLE 1 Effect of ginkgolides on the motor symptoms of ALS transgenic mice

Note: p < 0.05 compared to saline group.

TABLE 2 Effect of ginkgolides on the onset and survival time of ALS transgenic mice

Note: p < 0.05 compared to saline group.

3. Conclusion

The ginkgolide injection and the ginkgolide monomers, namely ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J and bilobalide, can improve the motor function of the ALS transgenic mice to different degrees, and have good improvement effect on female mice and male mice. As shown in the test results in Table 1, in the ginkgolide composition group (ginkgolide injection group, 14mg/kg) and the ginkgolide monomer group (ginkgolide group, ginkgolide B group, 14mg/kg) ALS transgenic mice of which the effective components comprise ginkgolide and ginkgolide, the exercise symptom score is remarkably higher than that of a normal saline group during the test period from 79 days to 109 days, the difference is statistically significant, the exercise symptom score is close to that of a positive control riluzole group (26mg/kg), and the tested ginkgolide composition, ginkgolide and ginkgolide B have good improvement effects on female mice and male mice. In the ginkgolide monomer group (ginkgolide A group, ginkgolide C group and ginkgolide J group, 14mg/kg) ALS transgenic mice, the exercise symptom score is obviously higher than that of a normal saline group in the test period of 79-95 days of age, the difference has statistical significance, the exercise symptom score is close to that of a positive control riluzole group (26mg/kg), and the tested ginkgolide A, ginkgolide C and ginkgolide J have good improvement effects on female mice and male mice.

The ginkgolide injection and the ginkgolide monomers, namely ginkgolide B and bilobalide, can also delay the morbidity of ALS transgenic mice to a certain extent. As shown in the test results in Table 2, the onset time of the ginkgolide composition group (ginkgolide injection group, 14mg/kg) and the ginkgolide monomer group (bilobalide, ginkgolide B group, 14mg/kg) ALS transgenic mice is obviously delayed, compared with the normal saline group, the difference is statistically significant, the onset time is similar to that of the positive control riluzole group (26mg/kg), and the tested ginkgolide composition, ginkgolide and ginkgolide B have good delaying effects on female mice and male mice. As shown in the test results in Table 2, the onset time of ALS mice in the ginkgolide monomer groups (ginkgolide A, ginkgolide C and ginkgolide J, 14mg/kg) is obviously delayed, and compared with the normal saline group, the difference is statistically significant (no sex statistics), and the onset time is close to that of the positive control riluzole group (26 mg/kg). Meanwhile, the ginkgolide injection and the ginkgolide monomers, namely ginkgolide B and bilobalide, can prolong the survival time of the ALS transgenic mice to a certain extent.

The animals in each group were tested before and after administration according to the test schedule, and no significant abnormality was observed. The ginkgolide disclosed by the invention is used for treating ALS and is good in safety.

In conclusion, the ginkgolide composition and the ginkgolide monomer can improve the motor function of the ALS transgenic mice and have good improvement effect on female mice and male mice. The ginkgolide composition and the ginkgolide monomer can delay the onset time, and the ginkgolide composition, the ginkgolide monomer ginkgolide B and the bilobalide have good delay effects on female mice and male mice. Animal test results show that the ginkgolide composition and the monomer thereof have good safety and can be further used for preventing, relieving or treating amyotrophic lateral sclerosis of mammals.

Claims

1. Use of ginkgo terpene lactones in the manufacture of a medicament for the prevention, alleviation or treatment of amyotrophic lateral sclerosis in a mammal.

2. Use according to claim 1, wherein the ginkgolide is optionally an effective amount of one of bilobalide or ginkgolide a or ginkgolide B or ginkgolide C or ginkgolide M or ginkgolide J or ginkgolide K or ginkgolide L or ginkgolide N or ginkgolide P or ginkgolide Q.

3. Use according to claim 1, wherein the ginkgolide is optionally an effective amount of two or more of bilobalide or ginkgolide a or ginkgolide B or ginkgolide C or ginkgolide M or ginkgolide J or ginkgolide K or ginkgolide L or ginkgolide N or ginkgolide P or ginkgolide Q.

4. The use of claim 3, wherein the ginkgolide comprises an effective amount of bilobalide and ginkgolide B.

5. The use of claim 3, wherein the ginkgolide comprises an effective amount of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C.

6. The use according to any one of claims 1 to 5, wherein the medicament is a pharmaceutical composition prepared from ginkgolides and/or pharmaceutically acceptable salts or hydrates thereof and/or isotopically labeled ginkgolides and pharmaceutical excipients or auxiliary components by a conventional method.

7. The use of claim 6, wherein the pharmaceutical composition is a tablet, a capsule, a granule, a pill, an injection, or a powder injection.

8. Use according to any one of claims 1 to 7, wherein the amyotrophic lateral sclerosis is familial amyotrophic lateral sclerosis.