阅读说明：本技术 盐酸丙美卡因及其衍生化合物在制备预防和/或治疗癫痫药物中的应用 (Application of proparacaine hydrochloride and derivative compound thereof in preparation of medicine for preventing and/or treating epilepsy ) 是由 周其冈 孟帆 韩峰 塔里布 周亚萍 朱明仪 张乔 张锦诚 于 2020-09-18 设计创作，主要内容包括：本发明公开了盐酸丙美卡因、其药学上可接受的盐、酯等衍生化合物或其前药在制备预防和/或治疗癫痫和/或癫痫并发症药物中的应用。本发明试验证明：造模小鼠给予盐酸丙美卡因(10mg/kg,腹腔注射),10分钟后癫痫波(epileptic spike)开始显著下降,并且自发性癫痫(spontaneous recurrent seizure)发作也被显著抑制；造模小鼠给予盐酸丙美卡因(10mg/kg,灌胃),癫痫小鼠的spike和SRS均被显著抑制；盐酸丙美卡因未造成显著的细胞损伤。(The invention discloses application of proparacaine hydrochloride, pharmaceutically acceptable salts, esters and other derivative compounds thereof or prodrugs thereof in preparing medicines for preventing and/or treating epilepsy and/or epilepsy complications. The test of the invention proves that: the procaine hydrochloride (10mg/kg, i.p.) administered to the model-making mice began to decrease significantly after 10 minutes (epileptic spike) and the spontaneous seizure (spontaneous seizure) was also significantly suppressed; when the procaine hydrochloride (10mg/kg, gavage) is given to the model-making mice, the spike and SRS of the epileptic mice are obviously inhibited; proparacaine hydrochloride caused no significant cellular damage.)

1. Application of compound shown as formula (I), pharmaceutically acceptable salt, ester or prodrug thereof in preparation of medicine for preventing and/or treating epilepsy and/or epilepsy complications

Wherein R1, R2 and R3 are alkyl groups with 1-4 carbon atoms; n is 1, 2 or 3.

2. The use according to claim 1,

the compound shown as the formula (I), pharmaceutically acceptable salts, esters or prodrugs thereof can be used for preparing products for preventing and/or treating epilepsy and/or epilepsy complications;

preparing a product that inhibits voltage-gated sodium channels;

preparing a product for inhibiting the sodium ion influx required for starting and conducting the pulse in the neuron cell.

3. The use according to claim 1,

the epileptic complications include reproductive hypofunction, neuroendocrine dysfunction and neuropsychiatric disorders.

4. A pharmaceutical composition comprising an effective amount of a compound of claim 1 or 2, a pharmaceutically acceptable salt thereof, or a prodrug thereof, in combination with one or more pharmaceutically acceptable pharmaceutical carriers.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical carrier comprises: diluent, excipient, adhesive, wetting agent, absorption enhancer, surfactant and lubricant.

6. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is in the form of a tablet, a capsule, an oral liquid, an injection, a powder, a liposome-encapsulated preparation, a paste, or a liquid for external use.

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to application of proparacaine hydrochloride and a derivative compound thereof in preparing a medicament for preventing and/or treating epilepsy.

Background

Epilepsy (Epilepsy) is a chronic brain disease that causes transient cerebral dysfunction due to sudden abnormal firing of cerebral neurons, and is suddenly initiated without any sign, and is one of the most common neurological diseases in all age groups. Epileptic seizures cause abnormalities in the neural circuits and activities in the brain, which cause the patient to suffer from cognitive, emotional, and mental disorders, not only seriously affect the normal work and life of the patient, but also shorten the average life span of the patient. Therefore, epilepsy is always an important social problem and global economic burden, and the pathogenesis and treatment strategy of epilepsy are also important concerns of researchers.

The clinical manifestations of seizures are mainly abnormal synchronized firing of neurons in the central nervous system, locally or in the whole brain area. The electrical activity balance between neurons is mainly formed by the mutual constraint of the activities of excitatory and inhibitory neurons, and the electrical balance of the neural network loop is affected by the increase of excitatory activity or the decrease of inhibitory activity in the brain. Ion channels are the core components responsible for the excitatory activity of the central nervous system (i.e., the conduction of neuronal action potentials) and the formation of neural circuits (i.e., the transmission of synaptic signals between neurons), and any genetic mutation in an ion channel may differentiate the normal functions of channel proteins, resulting in an imbalance in the electrical activity of the central nervous system, and finally induce abnormal synchronized discharges. Voltage-gated sodium channels produce action potentials in neurons, whose dysfunction can lead to hyperexcitable diseases such as hereditary epilepsy, chronic pain, etc.

Proparacaine hydrochloride is mainly used as a surface anesthetic at present and is applied to mucous membranes and corneal surfaces such as nasal cavities, throats, esophagus, trachea, urethra and the like and on eye corneas. In ophthalmology, the method is widely used for ophthalmology examination and various ophthalmology operations, such as cataract extraction; myopia by laser treatment; an tonometer measures intraocular pressure; suturing the operation and taking out foreign matters; conjunctival and corneal scraping, anterior chamber corneal examination, triple mirror examination, and the like.

The mechanism of the antiepileptic drug commonly used in clinic at present is to selectively act on a sodium channel, block the rapid release of a sodium ion-dependent action potential and regulate a voltage-dependent sodium ion channel. In addition, the medicines can also block calcium ion channels and regulate the activity of Na + -K + -ATP convertase, thereby achieving the effect of resisting convulsion. The development of a new antiepileptic drug is urgently needed.

Disclosure of Invention

Therefore, the invention provides the application of proparacaine hydrochloride and derivative compounds thereof in preparing the medicine for preventing and/or treating epilepsy.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides application of a compound shown as a formula (I), a pharmaceutically acceptable salt, an ester or a prodrug thereof in preparing a medicament for preventing and/or treating epilepsy and/or epilepsy complications

Wherein R1, R2 and R3 are alkyl groups with 1-4 carbon atoms; n is 1, 2 or 3.

The compound shown in the formula (I) is applied to the preparation of products for preventing and/or treating epilepsy and/or epilepsy complications, wherein the compound is shown in the formula (I), and pharmaceutically acceptable salts, esters or prodrugs of the compound; preparing a product that inhibits voltage-gated sodium channels; a product is prepared for inhibiting the influx of sodium ions required for the initiation and conduction of impulses in neuronal cells.

The epileptic complications include reproductive hypofunction, neuroendocrine dysfunction, and neuropsychiatric disorders.

The pharmaceutical composition comprises effective dose of the compound, the pharmaceutically acceptable salt thereof or the prodrug thereof, and one or more pharmaceutically acceptable pharmaceutical carriers.

The pharmaceutical composition as described above, wherein the pharmaceutical carrier comprises: diluent, excipient, adhesive, wetting agent, absorption enhancer, surfactant and lubricant.

The pharmaceutical composition is in the form of tablets, capsules, oral liquid, injection, powder, paste or external liquid medicine.

In the invention, the molecular formula of the proparacaine hydrochloride is C₁₆H₂₆N₂O₃HCl, molecular weight 330.86, solubility: DMSO:10mg/mL (30.2mM), Water:61mg/mL (184.4mM), Ethanol:<1mg/mL，(<1mg/ml refer to the product slide soluble or insoluble), the formula is shown below:

proparacaine hydrochloride is the hydrochloride form of proparacaine, a benzoic acid derivative with local anesthetic properties. Proparacaine hydrochloride stabilizes neuronal membranes by binding and inhibiting voltage-gated sodium channels, thereby inhibiting the influx of sodium ions required to initiate and conduct impulses within neuronal cells and resulting in loss of sensation.

In one embodiment of the present invention, the pharmaceutical composition is in the form of tablet, capsule, oral liquid, injection, powder, ointment or external liquid.

In the present invention, the term "prodrug" refers to a compound that is a drug precursor that, when administered, undergoes chemical transformation in vivo, either by metabolic or chemical processes (e.g., exposure to physiological pH or by enzymatic activity), releasing the active drug. The "prodrug" of the present invention may also include metabolic precursors of the compounds of the present invention, which may not be active when administered to a subject, but may be converted in vivo to a compound of formula (I) of the present invention or a salt and/or solvate thereof. Prodrugs can also be naturally occurring or chemically synthesized compounds.

The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

The terms "salt" and "pharmaceutically acceptable salt" refer to acid and/or base salts of a compound of formula (I) or a stereoisomer thereof, or a prodrug thereof, with inorganic and/or organic acids and bases, as well as zwitterionic (inner) salts, and also quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. Or by mixing a compound represented by the formula (I), or a stereoisomer thereof, or a prodrug thereof with a certain amount of an acid or a base as appropriate (e.g., an equivalent amount). These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization.

The tumors treated by the compounds of the present invention are not all tumors, but are specific tumors, such as tumors that are capable of apoptosis by a specific mechanism. According to the results of the test, the in vitro anti-tumor cell proliferation effect of the compound of formula (I) is caused by the inhibition of IKK beta. The compound (D6) of the formula (I) can directly bind to IKK beta and inhibit phosphorylation of downstream substrates thereof, thereby reducing the expression of a regulated gene (Bcl-2) and activating a Caspase family specific cleavage substrate PARP, thereby remarkably inducing apoptosis of tumor cells. In cell experiments, the proliferation inhibition activity of the compound of the formula (I) on various specific tumor cells can reach a single micromolar level.

In the present invention, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, and the like. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

The pharmaceutical compositions of the present invention are administered to a patient in need of such treatment by oral, nasal inhalation, rectal or parenteral administration. For oral administration, it can be made into conventional solid preparations such as tablet, powder, granule, capsule, etc., liquid preparations such as aqueous or oil suspension, or other liquid preparations such as syrup, elixir, etc.; for parenteral administration, it can be formulated into solution for injection, aqueous or oily suspension, etc.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient may be combined with one or more carriers and then formulated into the desired dosage form.

The invention has the following advantages:

the test of the invention proves that: propidocaine hydrochloride (10mg/kg, i.p.) was administered to the molded mice, spike began to drop significantly after 10 minutes, and SRS was also significantly inhibited; when the procaine hydrochloride (10mg/kg, gavage) is given to the model-making mice, the spike and SRS of the epileptic mice are obviously inhibited; proparacaine hydrochloride caused no significant cellular damage.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a brain wave effect graph of a mouse injected with 10mg/kg body weight proparacaine hydrochloride by acute intraperitoneal injection for the first time according to an embodiment of the present invention;

FIG. 2 is a graph showing the number of epileptic waves in a model mouse before and after administration, according to an embodiment of the present invention;

FIG. 3 is a statistical representation of the number of total epileptic waves and the number of epileptic seizures in a pre-and post-dose model mouse, provided by an embodiment of the present invention;

FIG. 4 is a statistical representation of the number of total epileptic waves and the number of epileptic seizures in a model mouse within 24 hours after administration provided by an embodiment of the present invention;

fig. 5 is a schematic diagram of a cytotoxicity test result of proparacaine hydrochloride according to an embodiment of the present invention.

Detailed Description

Other advantages and features of the present invention will become readily apparent to those skilled in the art from the following detailed description, wherein it is to be understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

Application of compound shown as formula (I), pharmaceutically acceptable salt, ester or prodrug thereof in preparation of medicine for preventing and/or treating epilepsy and/or epilepsy complications

Wherein R1, R2 and R3 are alkyl groups with 1-4 carbon atoms; n is 1, 2 or 3.

Specifically, the molecular formula of proparacaine hydrochloride provided in this example is C₁₆H₂₆N₂O₃HCl with a molecular weight of 330.86, the chemical formula is shown as the following formula (II):

the test of the invention shows that the proparacaine hydrochloride has better anti-epileptic effect in intraperitoneal injection and oral gavage, and has no obvious toxic or side effect. Proparacaine hydrochloride is the hydrochloride form of proparacaine, a benzoic acid derivative with local anesthetic properties. Proparacaine hydrochloride stabilizes neuronal membranes by binding and inhibiting voltage-gated sodium channels, thereby inhibiting the influx of sodium ions required in neuronal cells to initiate and conduct impulses, and resulting in loss of sensation.

Test example 1 intraperitoneal injection test of proparacaine hydrochloride of the invention to model mice

1. Experimental animals: c57BL/6J mice (8-10 weeks) were purchased from shanghai slaike laboratory animal accountant, all mice were housed according to standard regulatory guidelines and all experimental procedures on animals were performed according to the guidelines of the ethical committee on welfare of laboratory animals of the university of medical science, south kyo.

Reagents and consumables: pilocarpine hydrochloride (Pilocarpine hydrochloride, Pilocarpine) was purchased from Sigma, cat # P6503; scopolamine methyl nitrate (Scopolamine) was purchased from tcichemics under the reference number S0230; atropine methyl nitrate (Atropine methyl nitrate, Atropine) was purchased from Sigma, cat # SML 0732; terbutaline hemisulfate (Terbutaline hemisulfate salt, terbutalin) is available from Sigma under the cat number T2528; diazepam was purchased from hospitals.

In this example, the EEG brainwave detector used was purchased from Nihon Kohden, model EEG 1200C; screw electrodes were purchased from Plasticone under the trade designation E363-96-2.4-SPC, 6.5mm in length; straight electrodes were purchased from PlasticOne under the designation E363T-2-SPC, 7.5mm in length; electrode mounts were purchased from plasticcone, cat # MS 363; electrode converters were purchased from plasticcone under the designation SL 6C; electrode connector cables were purchased from PlasticOne, cat # 363-441/6, 3m in length.

2. Procedure of the test

Step one, constructing a spontaneous epilepsy model of a mouse by subcutaneous injection of Pilocarpine (Pilocarpine)

Pilocarpine is a cholinergic muscle agonist, which induces seizures when administered systemically, and the Pilocarpine model is currently the most widely used model of status epilepticus, as well as the spontaneous seizure model. The occurrence and development process of the medicine is highly similar to that of human temporal lobe epilepsy, has the same pathological basis, is resistant to most antiepileptic drugs, and is an ideal tool for researching the temporal lobe epilepsy.

The rating of epileptic seizure of mice is judged by adopting a Racine rating judgment standard: level 0: running normally without any other non-stressful reaction; first-stage: facial myoclonus, tremors such as blinking, beard beating, rhythmic chewing, etc.; and (2) second stage: adding rhythmic nod and forelimb clonus for the first-stage attack; third-stage: secondary attacks plus anterior limb myoclonus but no hind limb upstroke; and (4) fourth stage: three-stage hind limb vertical action accompanied by oblique walking, bilateral falling; and (5) fifth stage: the whole body is strong and straight, falls down, becomes unbalanced, rolls over and loses posture, and the limbs twitch. And judging the seizure to be severe at three levels or more, and using the seizure as a successful epilepsy model for subsequent experiments.

The body weight of the mice was measured, and the body weight of the mice was critical, and 22-28g of mice were most suitable, and all the mice were male mice. Scopolamine, Atropine, Terbutulin were intraperitoneally injected at a dose of 2mk/kg body weight, the injection time was recorded, and pilocarpine was injected 30 minutes later. Pilocarpine was injected subcutaneously at a dose of 280 mg/kg body weight, and then the shaking of mice was closely observed for 2-3 hours to evaluate the grade of seizures.

After 3h, mice were given intraperitoneal injection of diazepam to stop the epileptic seizure, the dose was 0.01 mg/mouse, and three or more levels of mice were selected to continue feeding and used for EEG electrode implantation and detection.

Step two, implanting an EEG electrode into the head of the epileptic model mouse

EEG (Electroencephalogram), brain wave monitoring is one of effective ways to study the electrical activity of the brain of an epileptic mouse, and EEG recording and analysis are started about 75 days after Pilocarpine modeling.

One week before electroencephalogram recording, under the anesthesia state of a mouse, five recording electrodes are directionally positioned by using a brain stereotaxic instrument: the left cortex and the right cortex are respectively 1, the prefrontal cortex is 1, the left hippocampus and the right hippocampus are respectively 1, and the rat is implanted in the cranium. The electrode is then attached to a connector mount which is fixed to the skull bone with dental cement. Mice were left unrestrained for 24-72 hours to recover from surgery before further procedures and long-term electroencephalographic monitoring began.

The mouse vertex connector base is connected to an EEG electroencephalogram detector through a converter and a connector cable, and digital electroencephalogram recording and analysis are carried out by using EEG2100 software. First, the electroencephalogram of each model mouse was recorded for 3 days before administration, generalized spontaneous seizures were defined as repetitive epileptiform seizure activity (>3Hz) lasting more than 3 seconds on all electrodes, and mice with spontaneous seizure brainwaves were selected as successful models of epilepsy. Then, the proparacaine hydrochloride with the anti-epileptic effect is intraperitoneally injected once a day for 3 consecutive days, the dosage is 10mg/kg of body weight, and the continuous electroencephalogram of the mouse is continuously recorded. Finally, the post-dose mouse electroencephalograms were recorded. During the recording, the mice were free to move and food and water were available ad libitum.

And (3) test results: 2.5 months after Pilocarpine modeling, mice developed stable epileptic brain waves (spike) and spontaneous seizure brain waves (SRS), mice were administered proparacaine hydrochloride (abbreviated D-5, 10mg/kg, i.p.), spike began to drop significantly after 10 minutes, and SRS was also significantly inhibited, as shown in FIGS. 1-3. Specifically, as shown in fig. 1, the frequency and amplitude of epileptic waves were significantly decreased 10 minutes after administration, 20 minutes after administration, and 30 minutes after administration, as compared to when epileptic waves were administered at the start of administration. As shown in fig. 2, the number of total epileptic waves within 24 hours after the administration was counted, indicating that the number of epileptic waves within 24 hours after the administration was significantly suppressed. As shown in fig. 3, the number of seizures within 24 hours after administration was counted, and the number of seizures within 24 hours after administration was significantly suppressed.

Test example 2 oral gavage test of proparacaine hydrochloride of the present invention on model mice

Step one, constructing a spontaneous epilepsy model of a mouse by subcutaneous injection of Pilocarpine (Pilocarpine)

Pilocarpine is a cholinergic muscle agonist, which induces seizures when administered systemically, and the Pilocarpine model is currently the most widely used model of status epilepticus, as well as the spontaneous seizure model. The occurrence and development process of the medicine is highly similar to that of human temporal lobe epilepsy, has the same pathological basis, is resistant to most antiepileptic drugs, and is an ideal tool for researching the temporal lobe epilepsy.

The rating of epileptic seizure of mice is judged by adopting a Racine rating judgment standard: level 0: running normally without any other non-stressful reaction; first-stage: facial myoclonus, tremors such as blinking, beard beating, rhythmic chewing, etc.; and (2) second stage: adding rhythmic nod and forelimb clonus for the first-stage attack; third-stage: secondary attacks plus anterior limb myoclonus but no hind limb upstroke; and (4) fourth stage: three-stage hind limb vertical action accompanied by oblique walking, bilateral falling; and (5) fifth stage: the whole body is strong and straight, falls down, becomes unbalanced, rolls over and loses posture, and the limbs twitch. And judging the seizure to be severe at three levels or more, and using the seizure as a successful epilepsy model for subsequent experiments.

The body weight of the mice was measured, and the body weight of the mice was critical, and 22-28g of mice were most suitable, and all the mice were male mice. Intraperitoneal injections of scopolamine, Atropine, and Terbutulin were given at 2mg/kg body weight, and the injection time was recorded, and pilocarpine was injected 30 minutes later. Pilocarpine was injected subcutaneously at a dose of 280 mg/kg body weight, and then the shaking of mice was closely observed for 2-3 hours to evaluate the grade of seizures.

After 3h, mice were given intraperitoneal injection of diazepam to stop the epileptic seizure, the dose was 0.01 mg/mouse, and three or more levels of mice were selected to continue feeding and used for EEG electrode implantation and detection.

Step two, implanting an EEG electrode into the head of the epileptic model mouse

EEG (Electroencephalogram), brain wave monitoring is one of effective ways to study the electrical activity of the brain of an epileptic mouse, and EEG recording and analysis are started about 75 days after Pilocarpine modeling.

One week before electroencephalogram recording, under the anesthesia state of a mouse, five recording electrodes are directionally positioned by using a brain stereotaxic instrument: the left cortex and the right cortex are respectively 1, the prefrontal cortex is 1, the left hippocampus and the right hippocampus are respectively 1, and the rat is implanted in the cranium. The electrode is then attached to a connector mount which is fixed to the skull bone with dental cement. Mice were left unrestrained for 24-72 hours to recover from surgery before further procedures and long-term electroencephalographic monitoring began.

The mouse vertex connector base is connected to an EEG electroencephalogram detector through a converter and a connector cable, and digital electroencephalogram recording and analysis are carried out by using EEG2100 software. First, the electroencephalogram of each model mouse was recorded for 3 days before dosing (pre-drug), generalized spontaneous seizures were defined as repetitive epileptiform seizure activity (>3Hz) lasting more than 3 seconds on all electrodes, and mice with spontaneous seizure brainwaves were selected as successful models of epilepsy. Then, the anti-epileptic effect of the present invention, proparacaine hydrochloride, was gavaged once daily for 3 consecutive days at a dose of 10mg/kg body weight, and the continuous electroencephalogram of the mice was continuously recorded. Finally, the post-dose mouse electroencephalograms were recorded. During the recording, the mice were free to move and food and water were available ad libitum.

2.5 months after Pilocarpine epileptogenesis modeling, the mice develop stable epileptic brain waves (spike) and spontaneous epileptic seizure brain waves (SRS), the modeled mice are given proparacaine hydrochloride (short for D-5, 10mg/kg, intragastric), and the spike and SRS of the epileptic mice are both obviously inhibited. As shown in fig. 4, the number of total epileptic waves and the number of epileptic seizures within 24 hours after the administration were counted, indicating that the number of epileptic waves and the number of epileptic seizures within 24 hours after the administration were significantly suppressed.

Test example 3 cytotoxicity test

In this example, PC12 cells were cultured, Cell Counting Kit-8 (CCK-8 for short) cytotoxicity test was performed, a Cell suspension was prepared, inoculated into a 96-well plate, subjected to adherent culture in an incubator at 37 ℃ and incubated with different concentrations (0.1. mu.g/ml, 1. mu.g/ml, 10. mu.g/ml, 100. mu.g/ml) of the drug for 24 hours, 10. mu.l of CCK-8 was added thereto and cultured for 4 hours, and then absorbance at 450nm was measured. Neither proparacaine hydrochloride nor the reference drug levetiracetam caused significant cellular damage, as shown in figure 5.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.