Combination medicines and their use for the treatment or prevention of epilepsy

Abstract

To provide pharmaceutical combinations containing Betula platyphylla sap extract for treating or preventing epilepsy while reducing a dosage of conventional anti-epileptic agents.SOLUTION: The present disclosure provides use of Betula platyphylla sap extract in producing compositions and medicines for treating or preventing epilepsy. The compositions further include other ingredients to enhance anti-epileptic effects. The compositions of the present disclosure have characteristics of high efficiency, safety, and convenience, and can effectively treat or prevent epilepsy seizure while reducing side effects of the anti-epileptic agents. The present disclosure also provides methods for treating or preventing epilepsy seizure with the compositions.SELECTED DRAWING: Figure 1A

Description

TECHNICAL FIELD

[0001] The present invention relates to a pharmaceutical combination for treating or preventing epilepsy and use thereof, and in particular to a pharmaceutical combination of birch sap extract and at least one drug and use thereof. BACKGROUND ART

[0002] The description of the background art contains useful information for understanding the present invention, and any information provided herein shall not be construed as background art or technology related to the present invention. Furthermore, any publication explicitly or implicitly cited shall not be construed as background art.

[0003] Epilepsy is a chronic disease of the nervous system, and it is the fourth most common neurological disorder after stroke, migraine and Alzheimer's disease, affecting approximately 70 million people worldwide. This disease is characterized by recurrent, spontaneous and unpredictable seizures caused by abnormal discharge of brain nerve cells, which mainly occurs in the cerebral cortex and hippocampus. The cause of epilepsy is related to congenital or acquired brain lesions, such as brain damage, infection, stroke, brain tumor, etc. However, the cause of onset in about 70% of epilepsy patients is unknown. Although most cases of epilepsy are idiopathic, it is generally believed that it is caused by excessive excitation of brain nerve cells due to the imbalance between the brain inhibitory nervous system (i.e., GABA system) and the excitatory nervous system (i.e., glutamic acid system).

[0004] Currently, the mechanisms of action of antiepileptic drugs that suppress epileptic seizures mainly involve drugs that directly suppress the excitation of brain nerve cells by blocking sodium channels (e.g., phenytoin, carbamazepine), drugs that suppress glutamate (e.g., lamotrigine, felbamate), or drugs that increase GABA (e.g., benzodiazepines, tiagabine, vigabatrin) to regulate the stability of brain nerve cells. However, although there are currently nearly 30 different epilepsy medications, about one-third of epilepsy patients have developed drug resistance to current medications, and the side effects of these drugs affect patients' adherence to their current treatments.

[0005] Currently, commonly used antiepileptic drugs are known to have many side effects. For example, the package insert for carbamazepine lists side effects such as drowsiness, loss of appetite, diarrhea, dizziness, headache, ataxia (dyscoordination), leukopenia, aplastic anemia, liver dysfunction, arrhythmia, and rash. The package insert for phenytoin lists side effects such as nausea, vomiting, heartburn, loss of appetite, double vision, nystagmus, ataxia (dyscoordination), gingival hyperplasia, hirsutism, facial rash, involuntary movements, lymphadenopathy, malformations, and rash. The usual treatment for side effects is to suppress them by changing the drug, reducing the dosage, or adding other medications. However, reducing the dosage makes epileptic seizures more likely to recur, causing both physical and mental distress to the patient.

[0006] Therefore, it is necessary to find and develop new drugs that are more effective and safer in the treatment of epilepsy, and medicinal plants are attracting attention because they are widely used in traditional and folk medicine to treat and prevent various neurological disorders (including epilepsy). In recent years, it has been proven that numerous plant-derived bioactive molecules produce antiepileptic effects through the influence of ion channels, GABA, or glutamic acid, and medicinal plants such as flavonoids, alkaloids, terpenoids, and cannabidiol have become research targets for antiepileptic drugs.

[0007] White birch (Betula platyphylla) belongs to the Betulaceae family and is distributed in regions such as Siberia, Finland, Hokkaido, and Mongolia. According to the "Compendium of Materia Medica," the bark of the white birch is used to treat arthritis, skin diseases, wounds, and to clear heat and detoxify. Currently, white birch is widely used in folk medicine to treat inflammatory diseases, including hepatitis, acute tonsillitis, pneumonia, chronic bronchitis, cholecystitis, urethral infections, and burns. Research has shown that white birch possesses numerous biological activities, such as anti-inflammatory, antioxidant, antiviral, anti-cancer, anti-fatigue, and neuroprotective effects, which are related to the triterpenoid betulin, the main active component of white birch. However, there is little research on the effects of white birch on the central nervous system, so a combination of medicines containing white birch extract is needed to treat or prevent epilepsy. [Overview of the project] [Problems that the invention aims to solve]

[0008] The technical problem that this invention aims to solve is to provide a combination of pharmaceuticals containing birch extract that can treat or prevent epilepsy and reduce the dosage of conventional antiepileptic drugs. [Means for solving the problem]

[0009] To address the above-mentioned problems and needs, the present invention provides applications for birch (Betula platyphylla) sap extract in the manufacture of pharmaceuticals for treating or preventing epilepsy.

[0010] In one embodiment, the birch sap extract of the present invention is collected from birch trees grown in Hokkaido, Japan.

[0011] In one embodiment, epilepsy is caused by damage to brain nerve cells. In another embodiment, epilepsy is caused by the activation of glial cells. In yet another embodiment, epilepsy is caused by an inflammatory response in the brain induced by inflammatory molecules. Furthermore, in yet another embodiment, the inflammatory molecules are selected from a group consisting of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group box 1 (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor-4 (TLR-4).

[0012] In one embodiment, the birch sap extract of the present invention is used in combination with at least one other antiepileptic drug to manufacture a drug for treating or preventing epilepsy. In another embodiment, the at least one other antiepileptic drug is a Western medicine formulation. Furthermore, in yet another embodiment, the at least one other antiepileptic drug is a Western medicine formulation selected from the group consisting of phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepine, thiagabine, and vigabatrin. In yet another embodiment, the at least one other antiepileptic drug is a Kampo medicine formulation or prescription. In yet another embodiment, the at least one antiepileptic drug is a Kampo medicine formulation or prescription selected from the group consisting of Gastrodia elata, Uncaria rhynchophylla, Citrus reticulata, Zhengan Xifeng Tang, and Tianma Gouteng Yin.

[0013] The present invention provides a method for producing birch sap extract, comprising the following steps. Step 1: Select a mature birch tree and cut the bark to make it easier for the sap to flow out; Step 2: Use a collector to gather the flowing sap; Step 3: Filter the sap at room temperature to remove bark fragments and other foreign matter; Step 4: A highly concentrated birch sap extract is obtained by removing most of the water from the sap using low-temperature concentration technology. The above steps may be increased or decreased depending on the extraction of specific components or other actual needs.

[0014] In one embodiment, the birch tree grew in Hokkaido, Japan. In another embodiment, the birch sap is collected in winter or spring. In yet another embodiment, the birch sap is collected in early spring.

[0015] The present invention also provides a combination of pharmaceuticals for treating or preventing epilepsy, comprising birch sap extract, at least one other antiepileptic drug, a pharmaceutically acceptable carrier and / or stabilizer. The dose of the other antiepileptic drug is less than that of the other antiepileptic drug administered alone. In one example, the at least one other antiepileptic drug is selected from the group of combinations of phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepine, thiagabine, and vigabatrin. In one example, the birch sap extract and the at least one other antiepileptic drug are administered to the patient at the same time, in the same order, or at intervals.

[0016] The various purposes, features, embodiments, and advantages of the present invention will become clear from the following detailed description of a better embodiment and the accompanying drawings. [Effects of the Invention]

[0017] The combination of pharmaceuticals containing birch extract according to the present invention can effectively treat or prevent epilepsy, and can also reduce the dosage of conventional antiepileptic drugs, thereby suppressing side effects. [Brief explanation of the drawing]

[0018] The features and advantages of the present invention will become further apparent from the following description of preferred embodiments in conjunction with the drawings, wherein like reference numerals refer to the same parts or members throughout the text.

[0019] [Figure 1A] It is a diagram showing the monosaccharide composition, which is a chemical property of birch sap extract (HCF02). [Figure 1B] It is a diagram showing the monosaccharide composition, which is a chemical property of birch sap extract (HCF02) [Figure 1C] It is a diagram showing the molecular weight distribution, which is a chemical property of birch sap extract (HCF02). [Figure 1D] It is a diagram showing the 1H NMR spectrum, which is a chemical property of birch sap extract (HCF02). [Figure 2A] It is a bar graph showing that pretreatment with birch sap extract reduces KA-induced epileptic behavior in animals. Data are presented as mean ± SEM (n=2~13 rats per group). * p<0.05 compared with the KA group; *** p<0.001 compared with the KA group. [Figure 2B] It is a bar graph showing that pretreatment with birch sap extract reduces KA-induced epileptic behavior in animals. Data are presented as mean ± SEM (n=2~13 rats per group). * p<0.05 compared with the KA group; *** p<0.001 compared with the KA group. [Figure 3A] It is a diagram showing that pretreatment with birch sap extract and betulin prevents KA-induced death of cerebral cortex and hippocampal neurons. Data are presented as mean ± SEM. * p<0.01 compared with the control group; *** p<0.001 compared with the control group; ♯ p<0.05 compared with the KA group; n=3 per group. [Figure 3B]It is a diagram showing that pretreatment with birch sap extract and betulin prevents KA-induced death of cerebral cortex and hippocampal neurons. Data are presented as mean ± SEM. * indicates p<0.01 compared with the control group; *** indicates p<0.001 compared with the control group; ♯ indicates p<0.05 compared with the KA group; n=3 for each group. [Figure 4A] It is a diagram showing that pretreatment with birch sap extract and betulin alleviates KA-induced degeneration of cerebral cortex and hippocampal neurons. Data are presented as mean ± SEM. *** indicates p<0.001 compared with the control group; ♯ indicates p<0.05 compared with the KA group; n=3 for each group. [Figure 4B] It is a diagram showing that pretreatment with birch sap extract and betulin alleviates KA-induced degeneration of cerebral cortex and hippocampal neurons. Data are presented as mean ± SEM. *** indicates p<0.001 compared with the control group; ♯ indicates p<0.05 compared with the KA group; n=3 for each group. MODE FOR CARRYING OUT THE INVENTION

[0020] The following description contains useful information for understanding the present invention, and any information provided herein shall not be construed as being related to the prior art or the claims of the present invention, and any publication explicitly or implicitly cited herein shall not be regarded as prior art.

[0021] The present invention is not limited to the materials, structures, processes, methods or structures of the specific examples recited herein. Therefore, although several alternatives are listed herein, all materials or methods with similar or equivalent effects can be applied to the implementation or examples of the present invention, and only the preferred materials and methods are described herein.

[0022] The following detailed description relating to the attached drawings is an illustrative example of the present invention and is not intended to indicate the only illustrative example of the invention. As used herein, the technical term “exemplary” means “one example, example, or case,” and it goes without saying that it may be better or superior to other possible exemplary examples. The detailed description includes specific parts and is provided for understanding the examples herein. Those skilled in the art will see that the examples herein can be carried out without using these specific parts.

[0023] As used herein, the following technical terms have the meanings set forth in their respective paragraphs. The technical terms used in this invention are for illustrative purposes only and are not intended to limit the use of specific embodiments of the invention.

[0024] Unless otherwise defined, the technical and scientific terms used herein are the same as those commonly understood by those skilled in the art. Generally, the nomenclature used herein is known and commonly used in the art in laboratory processes of animal pharmacology, pharmaceutical science, separation science, and organic chemistry. In the methods described herein, the operations can be performed in any order unless explicitly stated otherwise, such operations can be performed simultaneously. For example, the operation of performing X and the operation of performing Y can be performed simultaneously in a single operation, and the resulting method is within the scope of the claimed method.

[0025] The following non-restrictive initials are used herein: KA, kainic acid; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; HMGB1, high mobility group Box 1; IL-1R1, interleukin-1 receptor 1; CBZ, carbamazepine; CABA, gamma-aminobutyric acid; TLR-4, Toll-like receptor-4.

[0026] Unless explicitly indicated before or after the context, “one,” “one kind,” and “the” as used herein and in the claims include the plural. For example, “one kind of component” means one component or one or more components.

[0027] As used herein, those skilled in the art should understand the technical term “approximately” and it may vary to some extent depending on the context. As used herein, for measurable values ​​such as quantities and lengths of time, “approximately” means including variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% of the specified value. These variations are suitable for the disclosed methods. For this reason, in some embodiments, the numerical parameters in this specification and claims are approximations and will vary to obtain the expected characteristics by a particular method. In some embodiments, the numerical parameters are interpreted by applying general numerical tallying techniques according to the effective position described. Numerical values ​​in some embodiments of the present invention may have errors, which are due to the standard deviation in each test measurement.

[0028] In one embodiment, the technical term "co-administration" relating to the clinical trialer means administering to the clinical trialer, along with the birch sap extract of the present invention, other agents that treat or prevent the diseases considered herein. In some examples, the co-administered birch sap extract and other agents are administered separately, or any combination of them constitutes some single-treatment methods.

[0029] As used herein, “disease” refers to a type of health condition of a trial participant, where the participant is unable to maintain a stable internal state and the disease does not improve, resulting in a continued deterioration of the participant’s health.

[0030] As used herein, a participant's "medical condition" is a type of health state in which the participant's health is not better than when they are medically healthy, but they are able to maintain a stable internal state. Not treating the medical condition does not necessarily mean that the participant's health will deteriorate.

[0031] As used herein, the technical term “pharmaceutically acceptable” means a substance that does not negate the biological activity or properties of the useful compound in the present invention, such as a mediator or diluent. Furthermore, the substance is non-toxic, i.e., administering the substance to a test subject does not produce an unexpected biological reaction, nor do any components of the composition interact in a harmful manner.

[0032] As used herein, the technical terms “medicinal effective dose,” “therapeutic effective dose,” or “effective dose” of a compound refer to the amount of the compound that produces a beneficial effect on the patient to whom it is administered.

[0033] As used herein, the technical terms “investigator,” “individual,” and “patient” are interchangeable and include human or non-human mammals. Non-human mammals include, for example, livestock and pets, and include sheep, pigs, canids, felines, and rodents. In some embodiments, the investigator is human.

[0034] As used herein, the technical terms "prevention" or "prevention" mean that the time of administration of the drug or compound is before the subject develops a disease or medical condition, and that the onset of these symptoms is avoided or delayed.

[0035] As used herein, the technical term “treatment” means reducing the frequency or progression of a disease or condition experienced by an investigator by administering a drug or compound to the investigator.

[0036] Throughout the entirety disclosed in this invention, each aspect of the invention is expressed in the form of a range. The range format is for convenience and conciseness only and does not limit the scope of the invention. Therefore, the range should be considered as specifically all possible subranges and single numerical values ​​within those ranges. For example, the range description of 1 to 6 is considered to specify subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the single and partial numbers within that range are, for example, 1, 2, 2.7, 3, 4, 5, 5.3 and 6. All apply regardless of the range.

[0037] [About Birch Sap] The birch sap of this invention is a colorless or pale yellow transparent liquid collected and extracted from the bark and / or trunk of birch trees. Birch sap contains active substances such as betulin, polysaccharides, saponins, proteins, and minerals, and has relatively high medicinal value. In the theory of traditional Chinese medicine, birch sap belongs to the category of medicines that clear heat and detoxify, and the therapeutic effects and benefits disclosed to date are summarized below. Heat-clearing and detoxifying: Birch sap has a heat-clearing and detoxifying effect, removing heat and dampness from the body and alleviating symptoms such as fever, thirst, sore throat, and rash. Diuretic and anti-swelling: Birch sap can promote kidney excretion and increase urine output, thus removing excess fluid from the body and alleviating symptoms such as swelling. Anti-inflammatory and analgesic: Birch sap contains components with anti-inflammatory and analgesic properties, such as menthol and salicylic acid, which can alleviate pain and inflammation. Digestive aid: Birch sap can stimulate gastrointestinal motility and increase the secretion of digestive juices, thus improving gastrointestinal problems such as indigestion and diarrhea. Nutritional supplementation: Birch sap contains a variety of nutrients, such as vitamin C, B vitamins, iron, and potassium, which can replenish the body's essential nutrients and enhance its immunity and ability to fight disease.

[0038] In one example, birch sap is collected every winter or spring, with birch sap collected in early spring being preferable. In another example, birch sap is collected from birch trees aged 20 to 50 years. In yet another example, the birch trees are grown in Hokkaido, Japan.

[0039] In one embodiment, the birch sap extract of the present invention can be used in combination with one or more other pharmaceuticals for the treatment or prevention of epilepsy, or can be manufactured in combination with such pharmaceuticals. These pharmaceuticals include known compounds (e.g., commercially available compounds) that treat, prevent, or alleviate the symptoms of epilepsy.

[0040] In one embodiment, the present invention provides a method for treating or preventing epilepsy in a trial subject. This method involves administering a therapeutically effective dose of the birch sap extract of the present invention to the trial subject. In one embodiment, the trial subject is further administered at least one other drug effective in treating or preventing epilepsy. In another embodiment, the at least one other drug is a traditional Chinese medicine preparation or prescription such as Gastrodia elata, Uncaria rhynchophylla, Citrus monnieri, Zhengan Xifeng Tang, or Tianma Gouteng Yin, and the dosage of the traditional Chinese medicine preparation can be adjusted by a practitioner of traditional Chinese medicine according to conventional dosages. In another embodiment, the other drug is a traditional Chinese medicine preparation or prescription, which is compounded together with the birch sap extract. In another embodiment, the at least one other drug is a Western medicine preparation such as phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepine, thiagabine, or vigabatrin. The dosage of the Western medicine preparation can be adjusted by a physician according to the recommended dosage of each drug.

[0041] In one embodiment, the participants with epilepsy are those who have been diagnosed with epilepsy for the first time, or those who are taking at least one antiepileptic drug. In one embodiment, participants diagnosed with epilepsy for the first time are given a combination of birch sap extract and at least one antiepileptic drug, and the dose when administering the combination of at least one antiepileptic drug is lower than the dose when administering it alone. In another embodiment, when birch sap extract is given to participants who are taking at least one antiepileptic drug, the dose of the antiepileptic drug can be reduced.

[0042] The treatment method influences the combination of effective doses. Therapeutic formulations can be administered to patients before or after the onset of disease or symptoms. Furthermore, doses can be administered daily, in divided doses, alternating doses, or continuously. In addition, the dosage of the therapeutic formulation can be increased or decreased proportionally depending on the urgency of the treatment or prevention situation.

[0043] The present invention effectively treats or prevents epilepsy by administering the birch sap extract to patients in a known order, dosage, and duration. The effective amount of birch sap extract required for the therapeutic and / or preventive effect of epilepsy varies depending on many factors, such as the activity of the specific birch sap extract used; the time of administration; the excretion rate of the extract; the duration of treatment; other drugs, compounds, or substances used in combination with the extract; the disease or medical condition of the patient being treated, the patient's age, sex, weight, symptoms, general health status, and previous medical history; and similar factors familiar in the medical field. The dosage and other factors are adjusted to provide an excellent therapeutic response. For example, the dose may be administered in several divided doses daily, or the dose may be reduced in proportion to the urgency of the treatment situation.

[0044] The description of the birch sap extract of this invention primarily concerns its administration to humans in a manner consistent with medical ethics, but those skilled in the art should understand that the composition is generally suitable for various animals. Recombinations of the birch sap extract of this invention will be made known for administration to various animals, and skilled veterinary pharmacists can design and implement such recombinations through generality testing (if necessary). The subjects to whom the birch sap extract of this invention can be administered include, but are not limited to, humans and other primates, as well as non-mammalian mammals with commercial value, such as cattle, pigs, horses, sheep, cats, and dogs.

[0045] Humans and animals have different tolerances to the same drugs, and generally speaking, animal tolerance is stronger than human tolerance. When calculated per unit body weight or per unit surface area, animals use more drugs than humans. The amount used per unit body weight for animals and humans can be calculated by referring to the conversion table 1 below.

[0046] [Table 1]

[0047] Source: International Life Sciences Institute Taiwan, ILSI Taiwan

[0048] For example, using the rat model of the embodiment of the present invention, the birch sap extract of the present invention is administered orally in doses ranging from 3 mg / kg / dose to 20 mg / kg / dose, preferably 6 mg / kg / dose to 10 mg / kg / dose. After conversion using the above unit body weight dose conversion table, the dose of the birch sap extract to be administered to humans is 0.48 mg / kg / dose to 3.2 mg / kg / dose, preferably 0.96 mg / kg / dose to 1.6 mg / kg / dose. Doses of the birch sap extract of the present invention suitable for other animals can also be obtained using the above unit body weight dose conversion table or other unit body surface area conversion tables.

[0049] In some embodiments, the birch sap extract of the present invention is administered to patients in a dose range of one to five times or more daily. In other embodiments, the birch sap extract of the present invention is administered to patients in a dose range including, but not limited to, once daily, twice daily, three times daily, four times daily, every other day, every three days to a week, or once every two weeks. It will be apparent to those skilled in the art that the frequency of administration of each combination of compositions of the present invention will vary depending on the investigator, and this is influenced by many factors, including, but not limited to, age, the disease or condition being treated, sex, overall health, and other factors. Thus, the present invention should not be interpreted as being limited to any particular dose plan, and the attending physician should determine the exact dose and composition to be administered to the patient after considering all factors related to the patient.

[0050] In one embodiment, when the birch sap extract of the present invention is administered in combination with at least one other antiepileptic drug, the birch sap extract and at least one other antiepileptic drug can be administered to the test subject simultaneously. In another embodiment, the birch sap extract and at least one other antiepileptic drug can be administered to the test subject sequentially, before or after each other, or with an interval in between.

[0051] Oral administration: Liquid formulations used for oral administration are available in the form of solutions, syrups, or suspensions. These liquid formulations are manufactured by conventional methods using pharmaceutically acceptable additives, such as syrups or pharmaceutically acceptable preservatives.

[0052] Those skilled in the art will understand that, through experiments within the usual scope, equivalent items such as the specific sequence, method of implementation, claims, and examples described in this invention can be verified. These equivalent items are recognized as being within the scope of this invention and are further included in the claims. For example, modifications to reaction conditions such as reaction time, reaction state / volume and experimental reagents (e.g., solvents, catalysts), pressure, atmospheric pressure conditions, and reducing / oxidizing agents, as well as substitutes recognized in the art and experiments within the usual scope, are all within the scope of this invention.

[0053] Any numerical values ​​and ranges mentioned herein are provided for convenience and conciseness only and should not be interpreted as limitations on the scope of the invention. Therefore, all numerical values ​​and ranges included within these ranges are included within the scope of the invention. Furthermore, all numerical values ​​and ranges that fall within these ranges, whether upper or lower, are also assumed to be within the scope of the invention. All descriptions of ranges specifically disclose possible subranges and individual numerical values ​​within those ranges, and where appropriate, some integers within the numerical range are also considered to be included. For example, the description of the range from 1 to 6 is considered to be specifically disclosed as a subrange, and applies to all individual numbers within that range, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., regardless of the range, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6.

[0054] The following embodiments further illustrate aspects of the present invention. However, this does not limit the teachings or disclosures of the present invention described herein.

[0055] The present invention will be described with reference to the following embodiments. These embodiments are for illustrative purposes only, and the present invention is not limited to these embodiments, and includes all variations that become apparent through the teachings provided herein.

[0056] The birch sap extract exemplified in this invention is manufactured and / or tested by the following process.

[0057] [Birch sap extract] Birch sap extract is produced by collecting sap from birch (Betula platyphylla) in the following steps. Step 1: Select a mature birch tree and cut the bark to make it easier for the sap to flow out; Step 2: Collect the flowing sap with a collector to avoid external contamination; Step 3: Filter the sap at room temperature to remove bark fragments and other foreign matter; Step 4: A highly concentrated birch sap extract is obtained by removing most of the water from the sap using low-temperature concentration technology.

[0058] [Animal Experiments] Male Sprague-Dawley rats (SD, 150-200g) are purchased from BioLASCO TAIWAN Co., Ltd. and kept at the Fu Jen Catholic University Laboratory Animal Center. All animal experiments are authorized by the Fu Jen Catholic University Laboratory Animal Management Committee. Following the 3R rule, all experiments are conducted with the minimum number of animals necessary to produce reliable results while minimizing animal suffering as much as possible.

[0059] [Example 1 - Analysis of Birch Sap Components] Sap extracted from birch trees was analyzed using a monosaccharide analysis method. In the monosaccharide analysis of birch sap, the ratio of the three major monosaccharides in the birch sap extract—fructose, glucose, and fucose—was determined to be 27:17:10 (Figures 1A and 1B). Analysis by high-pressure size exclusion chromatography (HPSEC) revealed that the molecular weight of birch sap is approximately 1.29 kDa or less (Figure 1C). 1 The 1H NMR spectrum (Figure 1D) shows that there are two anomeric protons at δH5.13 and δH4.45. The signals at δH5.13 and δH4.45 show a bimodal pattern in a ratio of 100:124, with two monosaccharide residues and an α-anomeric structure, respectively. 3 J 1,2 =3.4Hz) and β-anomeric structure ( 3 J 1,2 It has a frequency of 7.7Hz. Based on the above analysis results, the amount of birch sap extract to be used in subsequent biological tests can be quantified.

[0060] [Example 2 - Animal epilepsy model induced by KA] The antiepileptic effect of birch sap extract is evaluated in an animal epilepsy induction model induced by kainic acid (KA). KA is a glutamate analog, and injection of KA into rats increases glutamate release and overactivates glutamate receptors, thus inducing progressive limbic seizures in rats. This leads to increased calcium ions in nerve cells, oxidative stress, and impaired glomerular function, resulting in nerve cell death in many areas of the brain, particularly the cerebral cortex and hippocampus. Furthermore, the induction of neuronal death and glial cell activation by KA is related to inflammatory responses mediated by inflammation-related molecules (e.g., interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high-mobility group proteins (HMGB1), interleukin-1 receptor 1 (IL-1R1), Toll-like receptor 4 (TLR-4)). Because these pathological changes induced by KA are similar to those of human epileptic behavior, KA-induced animal epilepsy models are widely used in the selection of novel compounds with antiepileptic activity.

[0061] After injecting KA into the peritoneal cavity of rats, epileptic behavior within 4 hours was analyzed. The method for evaluating seizure severity was based on the Racine Scale (1972), as shown in Table 2.

[0062] [Table 2]

[0063] [Example 3 - Evaluation of the effect of birch sap extract on KA-induced epileptic behavior in animals] Sprague-Dawley rats were administered different test substances and divided into the following groups. 1. Negative control group: Physiological saline. 2. KA group: 15 mg / kg / ip / single dose of kainic acid (KA). 3. Low-dose group of birch sap extract: 6 mg / kg of birch sap extract. 4. High-dose group of birch sap extract: 10 mg / kg of birch sap extract. 5. High-dose antiepileptic drug group: 100 mg / kg of carbamazepine (CBZ). 6. Antiepileptic drug composition group: Contains 6 mg / kg of birch sap extract and 50 mg / kg of carbamazepine (CBZ). 7. Low-dose antiepileptic drug group: 50 mg / kg of carbamazepine (CBZ).

[0064] Rats in the control group, birch sap extract group, antiepileptic drug group, and antiepileptic drug composition group were each administered physiological saline, birch sap extract, and / or carbamazepine at fixed times daily. After 7 days, excluding the negative control group, each group of rats was intraperitoneally injected with KA (15 mg / kg / ip / 1 dose). The dose of KA injection was based on previous studies. Epileptic behavior in rats was analyzed based on the Racine scale (1972) within 4 hours after intraperitoneal injection of KA, and the results are shown in Table 3.

[0065] Table 3 shows that pretreatment with birch sap extract reduces KA-induced epileptic behavior in animals.

[0066] [Table 3]

[0067] The above data are mean ± SEM. **, compared to the KA group, p<0.05; ***, compared to the KA group, p<0.001.

[0068] As the experimental results show, in the KA group (positive control group), 91% of rats given KA experienced seizures. The duration and score of the seizures were 66.6±6.4 minutes and 4.6±0.2 minutes, respectively. Compared to the KA group, pretreatment with oral administration of the birch sap extract of the present invention for 7 days extended the duration of KA-induced epileptic seizures and reduced the severity of KA-induced seizures (p<0.05; Figure 2A-2B). Furthermore, the high-dose group of birch sap extract (10 mg / kg) showed a good inhibitory effect on the severity of KA-induced epileptic seizures, and this effect is similar to that of the high-dose group of antiepileptic drugs (CBZ 100 mg / kg). Therefore, these results demonstrate that the birch sap extract of the present invention has an effect of preventing KA-induced epilepsy (Table 3). In other words, the birch sap extract of the present invention can be used as a substitute for antiepileptic drugs.

[0069] Furthermore, in the low-dose antiepileptic drug group, there was no statistically significant difference in the duration and severity of epileptic seizures in animals compared to the KA group. However, when used in combination with a low dose of birch sap extract, i.e., in the antiepileptic drug composition group (6 mg / kg birch sap extract + 50 mg / kg CBZ), the duration and severity of epileptic seizures in animals were clearly improved compared to the KA group (p<0.05; Figure 2A-2B), thus significantly improving the drawback that low-dose antiepileptic drugs cannot effectively suppress epileptic symptoms. Since the antiepileptic drug composition group produces an effect similar to that of the high-dose antiepileptic drug group in preventing KA-induced epilepsy, this result indicates that the combination of low-dose (6 mg / kg) birch sap and low-dose (50 mg / kg) CBZ can prevent KA-induced epileptic behavior in animals, and low-dose CBZ can further reduce the side effects caused by the antiepileptic drug.

[0070] Rat experiments have shown that the method provided by this invention can be used to prevent epileptic seizures, and has a clear preventive effect, particularly against epileptic symptoms that are difficult to control. When the birch sap extract of this invention is used alone, an antiepileptic effect equivalent to that of an antiepileptic drug (e.g., CBZ) is obtained. In other words, the birch sap extract can be used as a substitute for an antiepileptic drug. Furthermore, when the birch sap extract of this invention is used in combination with an antiepileptic drug, an antiepileptic effect can be achieved while reducing the dose of the antiepileptic drug (e.g., CBZ). As a result, whether the birch sap extract of this invention is used alone or in combination with an antiepileptic drug, the side effects of the antiepileptic drug can be reduced. Because the method provided by this invention has relatively low toxicity and side effects, it is a safe and effective method for preventing epilepsy.

[0071] [Example 4 - Pathological analysis of birch sap extract on brain nerve tissue] Following Example 3, experimental animals died 3 days after intraperitoneal injection of KA (15 mg / kg), and brain tissue was removed for biopsy and other correlational experiments were performed.

[0072] Three days after intraperitoneal injection of KA into rats, Nissl staining revealed a significant decrease in cerebral cortical and hippocampal CA1 / CA3 neurons in the KA group, indicating that pretreatment with birch sap extract and betulin can reduce the decrease in neuronal number induced by KA (p<0.05; Figures 3A and 3B).

[0073] Furthermore, Fluoro-Jade B (FJB) staining also showed an increase in the number of CA1 / CA3 degenerating cells in the cerebral cortex and hippocampus of the KA group, and this phenomenon was reduced by pretreatment with birch sap extract and betulin (p<0.05; Figures 4A and 4B). These preliminary results suggest that pretreatment with birch sap extract and / or betulin can prevent neuronal damage caused by KA.

[0074] Based on the results of the animal experiments described above, the birch sap extract of the present invention can extend the time that suppresses epileptic seizures induced by KA and reduce the severity of KA-induced epilepsy, thus confirming that the birch sap extract has an epilepsy-preventive effect. Therefore, birch sap has potential for use in the development of antiepileptic drugs and / or related products.

[0075] The following are illustrative examples, and their numbers should not be interpreted as indicating the importance of the designation.

[0076] Example 1 provides an application of birch sap extract in the manufacture of drugs for treating or preventing epilepsy.

[0077] Example 2 provides the same application as Example 1, and the birch sap extract is collected from birch trees grown in Hokkaido, Japan.

[0078] Example 3 provides the application of Example 1, wherein the epilepsy is caused by damage to brain nerve cells.

[0079] Example 4 provides the application of Example 1, wherein the epilepsy is caused by the activation of glial cells.

[0080] Example 5 provides the application of Example 1, in which epilepsy is caused by an inflammatory response in the brain due to inflammatory molecules.

[0081] Example 6 provides the application of Example 5, and the inflammatory molecule is selected from a group consisting of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group protein (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor 4 (TLR-4).

[0082] Example 7 provides the use of Example 1, further using at least one other antiepileptic drug in combination with the birch sap extract to manufacture a drug for treating or preventing epilepsy.

[0083] Example 8 provides the use of Example 7, wherein at least one other antiepileptic drug is a Western medicine formulation.

[0084] Example 9 provides the use of Example 8, wherein at least one other antiepileptic drug is a Western medicine formulation selected from the group of combinations of phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepine, thiagabine, and vigabatrin.

[0085] Example 10 provides the use of Example 7, wherein at least one other antiepileptic drug is a herbal medicine preparation or prescription.

[0086] Example 11 provides the use of Example 10, wherein at least one antiepileptic drug is a Kampo medicine preparation or prescription selected from the group consisting of a combination of Gastrodia elata, Uncaria rhynchophylla, Citrus latifolia, Zhengan Xifeng Tang, and Tianma Gouteng Yin.

[0087] Example 12 provides the use of Example 1, and the drug is used to treat or prevent epilepsy in humans.

[0088] Example 13 provides the use of Example 1, and the drug is used to treat or prevent epilepsy in non-human mammals.

[0089] Example 14 provides a method for producing birch sap extract, comprising the following steps. Step 1: Select a mature birch tree and cut the bark to make it easier for the sap to flow out; Step 2: Use a collector to gather the flowing sap; Step 3: Filter the sap at room temperature to remove bark fragments and other foreign matter; Step 4: A highly concentrated birch sap extract is obtained by removing most of the water from the sap using low-temperature concentration technology.

[0090] Example 15 provides the method of Example 14, and the birch tree was grown in Hokkaido, Japan.

[0091] Example 16 provides the method of Example 15, wherein the birch sap is collected in winter or spring.

[0092] Example 17 provides the method of Example 15, wherein the birch sap is collected in early spring.

[0093] Example 18 provides a combination of pharmaceuticals for treating or preventing epilepsy, comprising a birch sap extract prepared in any of Examples 14 to 17 and at least one other antiepileptic drug, wherein the dose of the other antiepileptic drug is less than the dose when administered alone.

[0094] Example 19 provides the pharmaceutical combination of Example 18, wherein the birch sap extract contains a pharmaceutically acceptable carrier and / or stabilizer.

[0095] Example 20 provides the drug combination of Example 18, wherein at least one other antiepileptic drug is selected from the group of combinations of phenytoin, carbamazepine, lamotrigine, felbamate, benzodiazepine, thiagabine, and vigabatrin.

[0096] Example 21 provides the combination of pharmaceuticals of Example 18, wherein at least one other antiepileptic drug is carbamazepine.

[0097] Example 22 provides the combination of pharmaceuticals of Example 18, wherein the birch sap extract and at least one other antiepileptic drug are administered to the patient simultaneously, sequentially, or at intervals.

[0098] While the present invention discloses certain embodiments, those skilled in the art can design other embodiments and variations, provided they do not deviate from the spirit and scope of the invention. The claims include variations that have the same effect as all embodiments. [Industrial applicability]

[0099] The combination of birch extract pharmaceuticals provided by this invention possesses high efficacy, safety, and convenience, and can effectively treat or prevent epilepsy while reducing the side effects that occur from the long-term use of conventional drugs.

Claims

1. The use of birch (Betula platyphylla) sap extract and carbamazepine for the manufacture of a drug for treating or preventing epilepsy, wherein the birch sap extract is obtained by filtering and concentrating birch sap collected from winter to spring at room temperature, and analysis by monosaccharide analysis shows that the ratio of fructose, glucose and fucose contained in the birch sap extract is 27:17:

10.

2. The use according to claim 1, characterized in that the birch sap extract is collected from birch trees grown in Hokkaido, Japan.

3. The use according to claim 1, characterized in that the epilepsy is caused by damage to brain nerve cells.

4. The use according to claim 1, characterized in that the epilepsy is caused by the activation of glial cells.

5. The use according to claim 1, characterized in that the epilepsy is caused by an inflammatory response in the brain due to inflammatory molecules.

6. The use according to claim 1 is characterized in that the inflammatory molecule is selected from a group consisting of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group protein (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor 4 (TLR-4).

7. (A) A first formulation of birch sap extract, wherein the concentration ratio of the three main monosaccharides contained in the birch sap extract is 27:17:10; and (B) a second formulation containing carbamazepine as the active ingredient, wherein the dose of carbamazepine in the second formulation is combined to be less than the dose when administered alone. This combination medicine is characterized in that it is a medicine for treating or preventing epilepsy.

8. The combination pharmaceutical product according to claim 7, characterized in that the components of the first formulation and the second formulation can be administered simultaneously, sequentially, or at intervals.

Images