A natural small molecule and its use in the preparation of a medicament

Abstract

The present application relates to the study of the biological activity of natural chemical small molecules and their use in the preparation of drugs, and relates to the use of 2-hydroxy-2-phenyl-malonamide in drugs.

Description

Technical Field

[0001] This invention belongs to the field of natural medicines, and relates to the bioactivity of a natural small molecule and the uses of the natural active molecule. Background Technology

[0002] Armillaria tabescens, scientifically known as *Armillariella pyrenoidosa*, possesses anti-inflammatory, antibacterial, and analgesic properties. It is significantly effective in treating gastritis, cholecystitis, cholangitis, appendicitis, and otitis media. It can promote bile secretion, nourish the liver and mucous membranes, boost metabolism, enhance liver and kidney function, and also has tonic and strengthening effects.

[0003] 2-hydroxy-2-phenylmalonamide (PHMA), also known as armillarisin B or false melleain B, has the following chemical structure (Formula 1). Armillarisin B is widely found in *Armillaria mellea* (commonly known as *Armillaria*), a fungus with both medicinal and edible properties. It has been reported that the content of armillarisin B in the fruiting body of *Armillaria mellea* reaches 0.25‰ of dry weight. Studies have shown that armillarisin B has a strong inhibitory effect on the mycelial growth of *Fusarium graminearum* (Shen Jin-Wen, Ma Bing-Ji, Li Wen, etc. Activity of armillarisin B in vitro against plantpathogenic fungi.[J]. Z. Naturforsch., C, J. Biosci., (11): 2-790.). However, its physiological activity in mammals, especially humans, has not been reported, and its clinical application value needs further research and development.

[0004]

[0005] Formula 1. 2-hydroxy-2-phenylmalonamide (PHMA) Summary of the Invention

[0006] The purpose of this invention is to develop the use value of natural compounds. 2-Hydroxy-2-phenylmalonamide (PHMA) is a secondary metabolite of *Gymnospermum*. Based on the bioactivity discovered during research, this invention further develops its uses in pharmaceuticals and other fields. The uses of PHMA and its derivatives in this invention include, but are not limited to, their use in the preparation of pharmaceuticals, and their application targets include all animals, especially vertebrates, including humans.

[0007] The medicines described in this invention refer to substances used for the prevention, treatment, and diagnosis of human diseases, and for the purposeful regulation of human physiological functions, and which have specified indications or therapeutic functions, usage, and dosage, including traditional Chinese medicine, chemical drugs, and biological products.

[0008] When the PHMA of the present invention is used as a drug, it can be added to the formulation as a pure product as an additive, or it can be added to the formulation as a component of other raw materials. The content can be fixed or within a range.

[0009] The PHMA and its derivatives of the present invention can be chemically synthesized compounds, biological extracts, or biological fermentation products.

[0010] In one specific embodiment, the PHMA of the present invention is chemically synthesized. There are various chemical synthesis methods, which have been disclosed and are well known to those skilled in the art.

[0011] In one specific embodiment, the PHMA of the present invention is a biological extract, such as *Bacillus thuringiensis*, and there are various methods for biological extraction, which have been disclosed.

[0012] In one specific embodiment, the PHMA of the present invention is a bio-fermentation product, and PHMA plays an active role as a component of the fermentation product.

[0013] PHMA and its derivatives exert their biological activity through routes such as injection and oral administration. Injection routes include intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, and other intracavitary injections. For ease of injection, PHMA and its derivatives can be formulated into pharmaceutical preparations, including liquid injections and dry powder injections. Oral routes include oral administration and gastrointestinal administration. For ease of oral administration, PHMA can be formulated into pharmaceutical preparations, including ordinary tablets, sustained-release tablets, controlled-release tablets, capsules, pills, granules, oral powders, oral liquid preparations, and lozenges. To better exert their biological activity, these formulations can be further subdivided, and the methods for various formulations are well known to those skilled in the art.

[0014] PHMA is slightly soluble in water. Solubility can be improved by adding a co-solvent, such as ethanol or dimethyl sulfoxide. The dissolution rate can also be increased by physical methods such as heating or stirring.

[0015] PHMA natural products and their derivatives can directly produce activity after entering the body; or participate in metabolism to form new active molecules; or inactive small molecules can participate in metabolism to form new active molecules; or active small molecules can participate in metabolism to form new inactive molecules. Specifically, the active form of this invention can be PHMA, which is hardly metabolized in the body and is generally secreted outside the body in its original form as excrement, such as sweat, urine, and feces; or only less than 50%, 40%, 30%, 20%, 10%, or 5% participates in human metabolism, and the metabolites can be amidation, esterification, deamination, oxidation, or reduction of PHMA to form new molecules.

[0016] This invention is the first to discover that PHMA has regulatory activity on the nervous system, specifically manifested in its use in maintaining sleep in animals or humans, promoting sedation and hypnosis, calming the nerves and nourishing the brain in animals or humans, and in the treatment and / or prevention of neurasthenia, anxiety, depression, insomnia, convulsions, or epilepsy. The sedative effect is characterized by reducing the frequency and duration of activity in animals. The hypnotic effect is characterized by shortening sleep onset time, accelerating sleep onset, or prolonging sleep duration.

[0017] The PHMA described in this invention can regulate the activity of the nervous system, meaning it has a sedative effect and can be used as a sedative drug. This includes reducing the activity level of animals during sleep, such as keeping the limbs or head still for extended periods, prolonging the duration of a single sleep episode, or extending the time of deep sleep.

[0018] PHMA has the activity of regulating the nervous system. It is a natural component with biological activity that acts on the nervous system and can be used to prevent or treat nervous system diseases and mental illnesses. It can also be used in the preparation of psychotropic drugs or nervous system drugs.

[0019] In one embodiment of the invention, PHMA can promote sleep in mammals, such as mice, by accelerating sleep onset and prolonging sleep duration.

[0020] In one embodiment of the invention, PHMA can reduce the activity level of mammals, such as mice, and can promote sleep in some mice, shortening the time it takes for them to fall asleep.

[0021] In one embodiment of the present invention, PHMA can induce a deep sleep state in mammals, such as mice. During sleep, there is no significant movement of the limbs and trunk, activity levels are reduced, the animal is in a resting state, and the duration of each sleep episode is prolonged. The resting state referred to in this invention means a state of complete stillness and rest, where there is no visually perceptible movement of the animal's limbs, head, tail, trunk, eyelids, mouth, etc., indicating a deep sleep state.

[0022] In one specific embodiment, PHMA is used alone as a drug; in another specific embodiment, it is used as a combination drug. In these embodiments, the dosage of PHMA can be 500 mg / kg body weight, or any dosage of 400 mg / kg body weight, 300 mg / kg body weight, 200 mg / kg body weight, 100 mg / kg body weight, 70 mg / kg body weight, 50 mg / kg body weight, 25 mg / kg body weight, or less than 500 mg / kg body weight. The final dosage is determined based on actual needs and safety.

[0023] In one specific embodiment, PHMA is administered in solution form at concentrations of 7.0‰, 5.0‰, 1.0‰, 0.5‰, 0.1‰, 0.05‰, and 0.01‰.

[0024] In one specific embodiment, PHMA was added to the mouse diet for a long-term feeding period of 30 days, and the body weight of the mice was not significantly different from that of the control group mice fed without PHMA. In another specific embodiment, PHMA was added to the drinking water of mice for a long-term drinking period of 30 days or more, and the body weight of the mice in the PHMA-added group was not significantly different from that of the control group mice fed without PHMA. The concentration of PHMA in the drinking water was 1.0‰, 0.5‰, 0.1‰, 0.05‰, 0.01‰ or lower.

[0025] In one specific embodiment, PHMA was added to the fish water of adult zebrafish at concentrations of 0.1‰, 0.05‰, and 0.01‰. The zebrafish survived normally for more than 30 days. It was observed that the zebrafish in the high-dose group had less activity than the zebrafish in the low-dose and no-PHMA groups.

[0026] In one specific embodiment, PHMA was added to the water used to raise adult zebrafish at a concentration of 1‰. The zebrafish showed a significant decrease in activity and frequently stopped swimming, remaining motionless. However, even when motionless, their pectoral fins could be observed to move or not move at all. After 24 hours, the water was replaced with water without PHMA, and the zebrafish returned to normal activity levels and survived for more than 30 days.

[0027] At 25°C, the solubility of PHMA in water is approximately 0.7 g / 100 g, and its solubility in aqueous solution varies considerably at different temperatures. In one specific embodiment, a co-solvent or solubilizer is added. The co-solvent can be ethanol, dimethyl sulfoxide, etc., or it can be directly dissolved in solvents such as ethanol or dimethyl sulfoxide.

[0028] The PHMA, its conjugates, and pharmaceutically acceptable salts of this invention exhibit regulatory activity of the nervous system in mice. As is well known to those skilled in the art, humans and mice share similar physiological characteristics, suggesting that PHMA may also exhibit a certain degree of regulatory activity of the nervous system in humans. Clinical research experience shows that the dosage used as a drug in experimental animals such as mice may differ significantly in humans, including substantial increases or decreases. Generally, the dosage per kilogram of body weight administered in humans is significantly lower than the dosage administered in experimental animals, for example, reduced to 1 / 2, 1 / 3, 1 / 5, 1 / 10, or 1 / 100 of the dosage administered in experimental animals.

[0029] Those skilled in the art can reasonably speculate on the use of PHMA or its pharmaceutically acceptable salts in the preparation of psychotropic or nervous system drugs. For example, they can reasonably speculate on the use of PHMA in the preparation of drugs for inducing and maintaining sleep in animals or humans, prolonging sleep time or deep sleep time, promoting sedation and hypnosis in animals or humans, calming the mind and improving brain function, and treating and / or preventing neurasthenia, anxiety, depression, insomnia, convulsions or epilepsy.

[0030] In one specific embodiment, PHMA or PHMA-containing extracts or compositions are used in the preparation of medicines for inducing and maintaining sleep in animals or humans, prolonging sleep time or deep sleep time, and treating and / or preventing insomnia.

[0031] In one specific embodiment, PHMA or PHMA-containing extracts or compositions are used in the preparation of sedative, hypnotic, calming, brain-boosting, therapeutic and / or preventative medications for neurasthenia, anxiety, depression, convulsions or epilepsy in animals or humans.

[0032] PHMA conjugates or their pharmaceutically acceptable salts can also be used as drugs to regulate the nervous system.

[0033] PHMA conjugates or their pharmaceutically acceptable salts can produce biological activity directly in vivo or after metabolism, including but not limited to sedation, hypnosis, and others. Their metabolism can occur in any organ in vivo or in a simulated in vitro environment.

[0034] The PHMA coupling compound or its pharmaceutically acceptable salt of the present invention, when used as a medicine, can replace PHMA, just like PHMA.

[0035] Implementation Methods

[0036] The following embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

[0037] Example 1: PHMA Direct Sleep Experiment

[0038] CD-1 (ICR) mice (approximately 30-35g body weight, SPF grade) were randomly divided into two groups of 10 mice each. The control and experimental groups received intraperitoneal injections of PHMA dissolved in physiological saline at 0 mg / kg and 70 mg / kg, respectively. Mice were placed in individual cages, and video monitoring was used to record their activity. The number of times the mice moved freely within 10 minutes (30-40 minutes after drug administration) was counted. Each movement was defined as a mouse turning 90-180 degrees and walking. The experiment was conducted in a quiet environment at 22℃.

[0039] The results showed that the number of spontaneous activities in the PHMA group was significantly reduced compared to the control group. Ten minutes after PHMA administration, the mice were significantly less active than the control group, rarely pacing back and forth between the two ends of the cage, and mostly lying still, exhibiting some looking around or sleeping behavior. Gently tapping the outside of the cage had no significant disturbance to the mice. This demonstrates that PHMA has a significant inhibitory effect on spontaneous activity in mice.

[0040] In addition, nine mice in the PHMA group remained still and fell asleep for more than 10 minutes at a time within one hour after drug administration, while only one mouse remained in a state of repeated brief periods of stillness. The mice in the control group were relatively more active.

[0041]

[0042] Mice in the PHMA group were tested for righting reflex after a single episode of sleep lasting more than 10 minutes following administration of the drug. When placed in a dorsal position, the mice were expected to immediately right themselves. If a mouse failed to right itself within 1 minute, the righting reflex was considered to have disappeared, indicating deep sleep. Five PHMA group mice remained asleep for more than 1 minute, while the other four righted themselves within 1 minute and remained asleep for a period. In contrast, all mice in the control group were alert and agile, and could right themselves immediately.

[0043] Example 2: PHMA prolongs sodium pentobarbital-induced sleep time

[0044] Kunming mice weighing 30-35g were divided into two groups of 10 each. The control group and the experimental group were administered physiological saline and PHMA dissolved in physiological saline (300ul), respectively, by gavage. After 50 minutes, all mice were injected intraperitoneally with sodium pentobarbital at a dose of 36 mg / kg body weight. The sleep time of each mouse was recorded.

[0045] The time interval between the disappearance and recovery of the righting reflex in mice was defined as sleep time. When mice were placed in a supine position, they would immediately turn over. If they could not turn over within 1 minute, the righting reflex was considered to have disappeared, and they had entered sleep. If they subsequently turned over, it was considered that they had awakened. The experiment was conducted in a quiet environment at 22℃.

[0046] The experimental results showed that PHMA could prolong the time it took for mice to fall asleep, from 17.73 minutes to 38.99 minutes, and the difference between the two groups was statistically significant (p<0.05). PHMA can prolong the sleep of mice.

[0047]

[0048] Example 3: Effect of PHMA on sleep at subthreshold doses of sodium pentobarbital

[0049] SPF-grade CD-1 (ICR) mice weighing 30-35g were divided into two groups of 10 mice each. The control group and the experimental group were intraperitoneally injected with saline and 300 μl of 5 mg / ml PHMA dissolved in saline (approximately equivalent to 50 mg / kg body weight), respectively. Twenty minutes later, sodium pentobarbital was injected intraperitoneally at a dose of 27 mg / kg body weight. The number of mice that entered a sleep state within 0.5 hours was recorded.

[0050] When mice are placed in a supine position, they will immediately turn over to their right position. If they cannot turn over within 1 minute, it is considered that the righting reflex has disappeared and they have entered a sleep state. The experiment was conducted in a quiet environment at 22℃.

[0051] Experimental results show that PHMA can increase the sleep rate of mice from 20% to 90%, and PHMA can promote sleep in mice and increase the sleep rate of animals taking subthreshold doses of sodium pentobarbital.

[0052]

Claims

Use of 1,2-hydroxy-2-phenylmalonamide or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of sedative and / or hypnotic drugs.

2. The use according to claim 1, characterized in that... The drug has a sedative effect and can reduce the frequency or duration of animal activity.

3. The use according to claim 1, characterized in that... The drug has a sedative effect and can prolong sleep time.

4. The use according to any one of claims 1-3, characterized in that, The drug is a formulation made of 2-hydroxy-2-phenyl-malondiamide or a pharmaceutically acceptable salt thereof, plus pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that, The drug is in the form of tablets, granules, capsules, oral solutions, or injections.

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