WO2024049179A1 - Electrophysiological evaluation method for drug efficacy by using in-vivo extracellular dorsal root ganglion recording method - Google Patents

Electrophysiological evaluation method for drug efficacy by using in-vivo extracellular dorsal root ganglion recording method Download PDF

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WO2024049179A1
WO2024049179A1 PCT/KR2023/012835 KR2023012835W WO2024049179A1 WO 2024049179 A1 WO2024049179 A1 WO 2024049179A1 KR 2023012835 W KR2023012835 W KR 2023012835W WO 2024049179 A1 WO2024049179 A1 WO 2024049179A1
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action potential
voltage
evaluation method
drug efficacy
sodium channel
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French (fr)
Korean (ko)
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김영범
김웅빈
손숙진
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주식회사 아이엔테라퓨틱스
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/48—Other medical applications
    • A61B5/4848—Monitoring or testing the effects of treatment, e.g. of medication
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/24—Detecting, measuring or recording bioelectric or biomagnetic signals of the body or parts thereof
    • A61B5/316—Modalities, i.e. specific diagnostic methods
    • A61B5/388—Nerve conduction study, e.g. detecting action potential of peripheral nerves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6846—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive
    • A61B5/6867—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive specially adapted to be attached or implanted in a specific body part
    • A61B5/6877—Nerve

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  • the present invention relates to an electrophysiological drug efficacy evaluation method using in-vivo extracellular dorsal root ganglion (DRG) recording, specifically, the degree of reduction in action potential normalized firing rate and/ Alternatively, it relates to a method of evaluating drug efficacy (pain inhibition effect) by confirming the degree of increase in the rate of change of the threshold value of standardized action potentials.
  • DRG dorsal root ganglion
  • Electrophysiological experimental techniques can be divided into patch-clamp recording method, cell-attached recording method, and extracellular recording method, and in the field of basic research, nervous system research is conducted. For this purpose, various electrophysiological techniques are used.
  • the present inventors applied the in vivo extracellular DRG recording method, which has been used only in basic research, to research for new drug development for the first time to electrophysiologically evaluate the effects of drugs in real time in living animals. A detailed method has been established to enable this, and the drug efficacy evaluation results actually derived using this method will be presented as a basis.
  • Non-patent Document 1 Neuron, Vol 109, Issue 9, Pages 1411-1582 (5 May 2021)
  • the purpose of the present invention is to apply an electrophysiological drug efficacy evaluation method using in-vivo extracellular nerve ganglion (DRG) recording to research on new drug development, inject a drug into a living animal, and electrophysiologically determine the effect of the drug.
  • the technical task is to provide an electrophysiological drug efficacy evaluation method using in-vivo extracellular nerve ganglion (DRG) recording to enable real-time evaluation.
  • the present invention discloses the following means.
  • the present invention uses in-vivo extracellular nerve ganglion (DRG) recording to use a voltage-dependent sodium channel inhibitor to selectively inhibit the activity of sodium channels, thereby causing the cell membrane potential to generate an action potential.
  • DRG in-vivo extracellular nerve ganglion
  • S1 vehicle to measure the normalized firing rate (%) of action potentials in the control firing rate section
  • S2 measuring the normalized firing rate (%) of action potentials in the drug firing rate section by administering a voltage-dependent sodium channel inhibitor
  • S3 statistically confirming the degree of inhibition of the action potential normalized firing rate measured in (S2) compared to the action potential normalized firing rate measured in (S1), and the activity in (S1) and (S2).
  • Potential normalized firing rate (%) provides a drug efficacy evaluation method characterized in that it is measured using the general formula 1 below.
  • B is the action potential generation frequency (Hz) generated in response to von Frey gram stimulation
  • B is each von Frey gram in the voltage-dependent sodium channel inhibitor firing rate section immediately after administration of vehicle or voltage-dependent sodium channel inhibitor. This is the frequency (Hz) of action potentials generated in response to stimulation.
  • the present invention uses in-vivo extracellular nerve ganglion (DRG) recording to use a voltage-dependent sodium channel inhibitor to selectively inhibit the activity of sodium channels, thereby causing the cell membrane potential to become an action potential.
  • DRG extracellular nerve ganglion
  • the electrophysiological drug efficacy evaluation method that evaluates the efficacy of drugs with a mechanism that reduces the frequency of action potentials by inhibiting or delaying reaching the threshold for inducing, (S1') vehicle Measuring the rate of change (normalized action potential threshold, %) of the threshold value of the normalized action potential by administering; (S2') measuring the normalized action potential threshold change rate (%) by administering a voltage-dependent sodium channel inhibitor; and (S3') statistically confirming the degree of increase in the rate of change in the threshold value of the standardized action potential measured in (S2') compared to the rate of change in the threshold value of the standardized action potential measured in (S1'). , the rate of change (normalized action potential threshold, %) of the normal
  • Y is the normalized action potential threshold change rate (%) after administration of vehicle or voltage-dependent sodium channel inhibitor
  • C is vehicle or voltage-dependent sodium channel
  • D is the minimum number of von Frey grams that generate an action potential before administration of the inhibitor
  • D is the minimum number of von Frey grams that generate an action potential after administration of a vehicle or voltage-dependent sodium channel inhibitor.
  • the electrophysiological drug efficacy evaluation method using in-vivo extracellular nerve ganglion (DRG) recording according to the present invention has the advantage of being directly applicable to in-vivo electrophysiological drug efficacy evaluation for new drug development. There is.
  • Figure 1 shows the intubation process for oral administration.
  • Figure 2 shows a schematic diagram of the in vivo extracellular DRG recording method according to the present invention.
  • Figure 3 illustrates an example of electrophysiological drug efficacy evaluation using the in vivo extracellular DRG recording method according to the present invention.
  • Figure 4 shows the results of electrophysiological drug efficacy evaluation using the in vivo extracellular DRG recording method according to the present invention.
  • Figure 5 shows the results of electrophysiological drug efficacy evaluation using the in vivo extracellular DRG recording method according to the present invention.
  • the numerical range includes the values defined in the range above. Every maximum numerical limit given throughout this specification includes all lower numerical limits as if the lower numerical limit were explicitly written out. Every minimum numerical limit given throughout this specification includes every higher numerical limit as if such higher numerical limit was clearly written. All numerical limits given throughout this specification will include all better numerical ranges within the broader numerical range, as if the narrower numerical limits were clearly written.
  • the inventors of the present invention confirmed the electrophysiological characteristics caused by mutations in conventional ion channels at the in-vivo level, thereby determining the method of drug administration and the use of the drug.
  • Research was conducted to solve the problem that criteria for determining effectiveness and detailed procedures and key elements for drug efficacy evaluation were not taken into consideration and therefore could not be directly applied to in-vivo electrophysiological drug efficacy evaluation for new drug development.
  • the present invention was completed by confirming that it can be evaluated.
  • the present invention discloses the following means.
  • the present invention uses in-vivo extracellular nerve ganglion (DRG) recording, wherein a voltage-dependent sodium channel inhibitor selectively inhibits the activity of the sodium channel, thereby causing the cell membrane potential to generate an action potential.
  • DRG extracellular nerve ganglion
  • a voltage-dependent sodium channel inhibitor selectively inhibits the activity of the sodium channel, thereby causing the cell membrane potential to generate an action potential.
  • S1 vehicle to measure the normalized firing rate (%) of action potentials in the control firing rate section
  • S2 measuring the normalized firing rate (%) of action potentials in the drug firing rate section by administering a voltage-dependent sodium channel inhibitor
  • S3 statistically confirming the degree of suppression of the action potential normalized firing rate measured in (S2) compared to the action potential normalized firing rate measured in (S1), and the activity in (S1) and (S2).
  • Potential normalized firing rate (%) provides a drug efficacy evaluation method characterized in that it is measured using the general formula 1 below.
  • B is the action potential generation frequency (Hz) generated in response to von Frey gram stimulation
  • B is each von Frey gram in the voltage-dependent sodium channel inhibitor firing rate section immediately after administration of vehicle or voltage-dependent sodium channel inhibitor. This is the frequency (Hz) of action potentials generated in response to stimulation.
  • in-vivo extracellular nerve bundle recording method refers to the action potentials generated in nerve bundle neurons and nerve bundle filament areas in anesthetized mice using microglass pi. This refers to a method of recording from outside the cell using a pet.
  • the term “voltage-dependent sodium channel inhibitor” refers to a low-molecular-weight compound that inhibits the activity of a channel that serves as a passage for the movement of sodium ions in a specific range of membrane voltage.
  • frequency of occurrence refers to the degree of frequency of occurrence of action potentials.
  • control firing rate refers to the 2 firing rate generated through von Frey stimulation at 30, 60, 90, and 120 minutes after administration of the vehicle, respectively. This is the action potential generation section for seconds and refers to the vehicle effect section for deriving the difference value compared to the time immediately before vehicle administration.
  • drug firing rate section refers to the 2-second period generated through von Frey stimulation at 30, 60, 90, and 120 minutes after drug administration, respectively. This is the action potential generation section and refers to the drug effect section for deriving the difference value compared to the time immediately before drug administration.
  • normalized firing rate (%) refers to the change in action potential firing rate that occurs after administering the drug compared to before administering the drug in response to von Frey stimulation, converted into a percentage. It means one thing.
  • the drug efficacy evaluation method may satisfy the following conditions.
  • Condition 2 In General Formula 1 above, the action potential normalized firing rate measured in (S2) is smaller than the action potential normalized firing rate measured in (S1), and the difference is statistically significant.
  • a statistically significant case means a case that passes the significance level specified through the statistical test set below.
  • the null hypothesis of statistics is "the action potential normalized firing rate measured in (S2) is not smaller than the action potential normalized firing rate measured in (S1).”
  • the alternative hypothesis is “The action potential normalized firing rate measured in (S2) is smaller than the action potential normalized firing rate measured in (S1).” am.
  • the significance level is the universal standard p-value ⁇ 0.05. If the above conditions are met, the null hypothesis is rejected and the significance level is passed. The generally set values are P ⁇ 0.05, P ⁇ 0.01, P ⁇ 0.001
  • the level of significance was divided into three levels.
  • the experimental results are expressed as mean ⁇ SEM, and the results obtained from the control group (S1) and the experimental group (S2) were subjected to a post-hoc pairwise comparison using two-way ANOVA. ) was performed using the statistical analysis program provided by Prism.
  • the voltage-dependent sodium channel inhibitor may be a compound represented by the following formula (1), but is not limited thereto.
  • the compound represented by Formula 1 can selectively inhibit the activity of the Nav1.7 sodium channel, but is not limited thereto.
  • the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor can be administered in an amount of 1 mg/kg to 100 mg/kg, but is not limited thereto.
  • 30 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor can be administered, but is not limited thereto.
  • X, A, and B when 30 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, is administered, in Formula 1, X, A, and B may be as follows, but are not limited thereto. .
  • the drug efficacy evaluation method involves fixing the vertebrae using a spinal cord clamper to perform extracellular recording in a specific target area of the spinal cord after anesthetizing a rodent before step (S1). It may include steps, but is not limited thereto.
  • rodent means related to mouse or rat.
  • the von Frey gram may be 1 to 100 g, specifically, may be any one or more selected from the group consisting of 1 g, 2 g, 4 g, 10 g, and 100 g, and is limited thereto. no.
  • the present invention uses in-vivo extracellular nerve ganglion (DRG) recording to use a voltage-dependent sodium channel inhibitor to selectively inhibit the activity of sodium channels, thereby increasing the cell membrane potential.
  • DRG extracellular nerve ganglion
  • Y is the normalized action potential threshold change rate (%) after administration of vehicle or voltage-dependent sodium channel inhibitor
  • C is vehicle or voltage-dependent sodium channel
  • D is the minimum number of von Frey grams that generate an action potential before administration of the inhibitor
  • D is the minimum number of von Frey grams that generate an action potential after administration of a vehicle or voltage-dependent sodium channel inhibitor.
  • normalized action potential threshold (%) refers to the minimum stimulation required to induce an action potential after administration of the drug, based on before administration of the drug. This means that the change value has been converted into a percentage.
  • the drug efficacy evaluation method may satisfy the following conditions.
  • Condition 4 In the above general formula 2, the rate of change of the threshold value of the standardized action potential measured in (S2') is greater than the rate of change of the threshold value of the standardized action potential measured in (S1'), and the difference is statistically This is a significant case.
  • a statistically significant case means a case that passes the significance level specified through the statistical test set below.
  • the null hypothesis of statistics is "The rate of change in the threshold value of the standardized action potential measured in (S2') is not greater than the rate of change in the threshold value of the standardized action potential measured in (S1')."
  • the alternative hypothesis is "The rate of change in the threshold value of the standardized action potential measured in (S2') is greater than the rate of change in the threshold value of the standardized action potential measured in (S1').”
  • the significance level is the universal standard p-value ⁇ 0.05. If the above conditions are met, the null hypothesis is rejected and the significance level is passed.
  • the generally set values are P ⁇ 0.05, P ⁇ 0.01, P ⁇ 0.001
  • the level of significance was divided into three levels.
  • the experimental results are expressed as mean ⁇ SEM, and the results obtained from the control group (S1') and the experimental group (S2') were compared pairwise through a post-hoc test using two-way ANOVA ( Pairwise comparison) was performed using the statistical analysis program provided by Prism.
  • the voltage-dependent sodium channel inhibitor may be a compound represented by the following formula (1), but is not limited thereto.
  • the compound represented by Formula 1 can selectively inhibit the activity of the Nav1.7 sodium channel, but is not limited thereto.
  • the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor can be administered in an amount of 1 mg/kg to 100 mg/kg, but is not limited thereto.
  • the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor can be administered at 30 mg/kg, but is not limited thereto.
  • Y, C and D when administering 30 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, in Formula 2, Y, C and D may be as follows, but are not limited thereto. .
  • the drug efficacy evaluation method uses a spinal cord clamper to perform extracellular recording in a specific target area of the spinal cord after anesthetizing a rodent before the (S1') step. It may include a fixing step, but is not limited thereto.
  • rodent means related to mouse or rat.
  • mice were used.
  • the breed of mice used was ICR, 10 mice were used, the gender was male, the target age was 5 to 7 weeks, the initial weight was 25 to 40 g, and they were supplied by Coretech Co., Ltd.
  • Anesthesia was administered by intraperitoneal injection of urethane at a dose of 1.2 g/kg using a sterilized 1cc syringe (26 gage) to prepare for jugular cannulation for exposure of the nerve bundle ganglion (DRG).
  • Example 2 Intubation for oral administration
  • Intubation for oral administration was performed to secure the drug administration route, as shown in Figure 2.
  • medical polyethylene tubing for intubation for oral administration was PE 10 with an inner diameter of 0.011 inches and an outer diameter of 0.024 inches.
  • PE 10 polyethylene tubing
  • zonde for oral administration mouse, 1.2 in 10
  • the needle for oral administration was removed again, leaving only the remaining one.
  • Example 3 Exposure of nerve bundle ganglia (DRG) for electrophysiological recordings
  • a heating pad was placed on the ventral side of the mouse to maintain body temperature without direct contact with the cold floor, and eye ointment was applied liberally to both eyes to prevent them from drying out.
  • DRG spinal cord and nerve bundle ganglion
  • Example 4 In vivo ( in-vivo ) Extracellular ganglion (DRG) recordings.
  • a fine glass pipette with a diameter of approximately 1 to 2 ⁇ m was used.
  • the resistance was prepared by making a glass pipette with a resistance of about 10 to 20 M ⁇ using a puller, and inside the pipette was an artificial cerebrospinal fluid (ACSF) solution (124 mM NaCl, 1.3 mM MgSO 4 , 3 mM KCl, 1.25 mM NaH 2 PO 4 , 26mM NaHCO 3 , 2.4mM CaCl 2 , and 10mM glucose) were filled and used for recording.
  • ACSF cerebrospinal fluid
  • the dura surrounding the nerve bundle ganglion (DRG) was finely torn using a fine glass pipette to prevent damage to the tip of the pipette during the extracellular recording method, and artificial cerebrospinal fluid (artificial cerebrospinal fluid) was used.
  • artificial cerebrospinal fluid artificial cerebrospinal fluid
  • action potential (AP) generation was observed by sweeping the knee or sole of the mouse with a brush to check whether there was a response to stimulation.
  • nerve bundle ganglia were stimulated in response to specific grams (g) of von Frey stimulation (1 g, 2 g, 4 g, 10 g, 100 g; three stimulations for 5 seconds at 5-second intervals).
  • g specific grams
  • g Changes in the action potential (AP) firing rate generated from neurons (firing rate test; FT) and changes in the size of the minimum stimulus (von Frey) required to induce an action potential (AP) (threshold test) , threshold test (TT) was observed (see Figure 3).
  • the evaluation drug is a voltage-dependent sodium channel inhibitor, a compound represented by the following formula (iN1011-N17, 30 mg/kg; oral administration), which inhibits the activity of a specific subtype (Nav1.7) of sodium channels among various voltage-dependent sodium channels.
  • a drug was used that reduces the frequency of action potential (AP) occurrence by selectively inhibiting or delaying the cell membrane potential from reaching the threshold for inducing an action potential (AP).
  • the first parameter for drug efficacy evaluation was the change in firing rate (FT), which used the frequency (Hz) of action potentials (AP) generated by von Frey stimulation.
  • FT firing rate
  • AP action potentials
  • the second parameter for drug efficacy evaluation is the change (TT) in the size of the minimum stimulus (von Frey), and the minimum von Frey gram (g) stimulus that generates an action potential (AP) was used.
  • TT change in the size of the minimum stimulus
  • g minimum von Frey gram
  • AP action potential
  • (1 g, 2 g, 4 g, 10 g, 100 g) Represents the action potential (AP) generation frequency (Hz) generated in response, and B is 30, 60, 90, 120 after vehicle or drug administration.
  • the group administered iN1011-N17 a voltage-dependent sodium channel inhibitor (formula 1 compound, experimental group) was different from the group administered the vehicle (control group) in all subjects. Regardless of the intensity of stimulation, a lower frequency of action potential (AP) occurrence in the nerve bundle ganglion (DRG) was recorded after drug administration than before administration. This was clearly different compared to the control group, and the difference was confirmed to be a statistically significant decrease.
  • the drug effect in this experimental group lasted up to 2 hours after drug administration, and it was confirmed that the degree to which the frequency of action potential (AP) was suppressed gradually strengthened over time.
  • the present invention overcomes the difficulty in securing proof of concept (POC) and proof of mechanism (POM) of the developed drug action in the process of developing a drug with a mechanism to suppress pain by changing the activity of ion channels. It is judged to be a reliable alternative method.
  • C represents the minimum number of von Frey grams that generate an action potential before administration of the vehicle or drug
  • D represents the minimum number of von Frey grams that generate an action potential after administration of the vehicle or drug.
  • Y is the rate of change (normalized action potential threshold, %) of the normalized action potential (AP) threshold for deriving the result of Figure 5
  • C is the activity before vehicle or drug administration.
  • D is the minimum number of von Frey grams that generate an action potential (AP)
  • AP is the minimum number of von Frey grams that generate an action potential (AP) at 30, 60, 90, and 120 minutes after vehicle or drug administration. It is the number of grams.
  • the Y value which is the normalized action potential threshold (%) change rate of the normalized action potential (AP) threshold after administration of the vehicle or drug, was derived.
  • the group administered iN1011-N17 had a larger effect after drug administration, unlike the group administered the vehicle (control group). It was confirmed that action potentials were generated in the nerve bundle ganglion (DRG) only through stimulation.
  • DRG nerve bundle ganglion
  • the present invention overcomes the difficulty in securing proof of concept (POC) and proof of mechanism (POM) of the developed drug action in the process of developing a drug with a mechanism to suppress pain by changing the activity of ion channels. It is judged to be a reliable alternative method.

Abstract

Disclosed in the present invention is an electrophysiological evaluation method for drug efficacy by using an in-vivo extracellular dorsal root ganglion (DRG) recording method.

Description

인비보 세포외 신경다발절 기록법을 이용한 전기생리학적 약효평가법Electrophysiological drug efficacy evaluation method using in vivo extracellular nerve bundle recording method
본 발명은 인비보(in-vivo) 세포외(extracellular) 신경다발절 (dorsal root ganglion; DRG) 기록법을 이용한 전기생리학적 약효평가법에 대한 것으로서, 구체적으로는 활동전위 표준화된 발화율의 감소 정도 및/또는 표준화된 활동전위의 역치값의 변화율의 증가 정도의 확인을 통해 약효(통증의 억제 효과)를 평가하는 방법에 관한 것이다.The present invention relates to an electrophysiological drug efficacy evaluation method using in-vivo extracellular dorsal root ganglion (DRG) recording, specifically, the degree of reduction in action potential normalized firing rate and/ Alternatively, it relates to a method of evaluating drug efficacy (pain inhibition effect) by confirming the degree of increase in the rate of change of the threshold value of standardized action potentials.
이온채널 (ion channels)에 대한 연구가 활발해지고 이에 대한 중요한 생리적인 기능이 밝혀짐에 따라서 이온채널 매개의 질병을 치료하기 위한 시도가 점차 확대되어 가고 있다.As research on ion channels becomes more active and their important physiological functions are revealed, attempts to treat ion channel-mediated diseases are gradually expanding.
이온채널을 표적으로 한 치료제를 개발하기 위해서는 전기생리학 실험 장비 및 기술이 필수적이며, 그 방법에는 여러가지가 있을 수 있다.In order to develop therapeutic agents targeting ion channels, electrophysiology experimental equipment and technology are essential, and there are many different methods.
전기생리학적 실험 기법은 패치클램프 기록법 (patch-clamp recording method), 세포 접촉 기록법 (cell-attached recording method), 세포외 기록법 (extracellular recording method) 등으로 구분될 수 있고, 기초연구 분야에서는 신경계 연구를 위한 목적으로 여러가지 전기생리학 기법을 이용하고 있다.Electrophysiological experimental techniques can be divided into patch-clamp recording method, cell-attached recording method, and extracellular recording method, and in the field of basic research, nervous system research is conducted. For this purpose, various electrophysiological techniques are used.
제약산업 분야에서는 전기생리학 플랫폼 (electrophysiological platform)을 도입하여 신약 개발 연구에 사용하는 것이 초기 단계이기 때문에 제약사 대부분이 현재까지 다양한 전기생리학 실험 기법을 구현하여 신약 개발에 이용하기는 어려운 실정이다.In the pharmaceutical industry, the introduction of electrophysiological platforms and their use in new drug development research is at an early stage, so it is difficult for most pharmaceutical companies to implement various electrophysiological experiment techniques and use them for new drug development.
통증 (pain)을 포함한 많은 신경계 질환 (neurological diseases) 치료제를 개발하기 위해서는 단순한 세포 수준에서부터 보다 복잡한 조직 수준, 그리고 살아있는 개체 수준까지 여러 단계에 거쳐 연구가 수행되어야 한다.In order to develop treatments for many neurological diseases, including pain, research must be conducted at multiple levels, from simple cellular levels to more complex tissue levels and living organism levels.
특히 이온채널과 관련이 있는 신경계 질환의 경우 전기생리학 기법을 이용한 연구가 필수적이라고 할 수 있지만, 화합물의 인비트로 (in-vitro) 활성 평가에만 전기생리학 기법을 사용하는 수준에 그치는 실정이다.In particular, in the case of neurological diseases related to ion channels, research using electrophysiological techniques is essential, but the current situation is that electrophysiological techniques are only used to evaluate the in-vitro activity of compounds.
최근 한 논문을 보면, 마우스에서 전압 의존성 소디움 채널 1.7 (voltage-dependent sodium channel 1.7; Nav1.7)의 돌연변이를 유도하고, 이러한 돌연변이가 나타내는 기능적인 변화를 신경다발절(DRG)에서 인비보 세포외 신경다발절 기록법(in vivo extracellular DRG recording method)을 이용하여 전기생리학적으로 확인한 바 있다.In a recent paper, mutations in voltage-dependent sodium channel 1.7 (Nav1.7) were induced in mice, and the functional changes exhibited by these mutations were measured in vivo extracellularly in the nerve bundle ganglia (DRG). It was confirmed electrophysiologically using the in vivo extracellular DRG recording method.
그러나, 이 연구는 단지 이온채널의 변이에 의해 나타나는 전기생리학적인 특성을 인비보(in-vivo) 수준에서 확인 한 것이기에 약물의 투여 방법, 약물의 효과를 판단하기 위한 기준, 그리고 약효 평가를 위한 세부 절차와 핵심 요소 등은 고려되어 있지 않아서 신약 개발을 위한 인비보 전기생리학적 약효 평가에는 그대로 적용될 수 없다는 문제가 있다.However, since this study only confirmed the electrophysiological characteristics caused by mutations in ion channels at the in-vivo level, the method of drug administration, standards for judging drug effectiveness, and details for evaluating drug efficacy were determined. There is a problem that it cannot be directly applied to in vivo electrophysiological drug efficacy evaluation for new drug development because the procedures and key elements are not taken into consideration.
본 발명자들은 기초 연구에만 사용되어져 온 인비보 세포외 신경다발절 기록법(in vivo extracellular DRG recording method)을 최초로 신약 개발을 위한 연구에 적용하여, 전기생리학적으로 살아있는 동물로부터 약물의 효과를 실시간으로 평가할 수 있도록 세부적인 방법을 수립하였고, 이 방법을 이용하여 실제 도출한 약효 평가 결과를 근거로 제시하고자 한다.The present inventors applied the in vivo extracellular DRG recording method, which has been used only in basic research, to research for new drug development for the first time to electrophysiologically evaluate the effects of drugs in real time in living animals. A detailed method has been established to enable this, and the drug efficacy evaluation results actually derived using this method will be presented as a basis.
[선행기술문헌][Prior art literature]
[비특허문헌][Non-patent literature]
(비특허문헌 1) Neuron, Vol 109, Issue 9, Pages 1411-1582 (5 May 2021)(Non-patent Document 1) Neuron, Vol 109, Issue 9, Pages 1411-1582 (5 May 2021)
본 발명의 목적은 인비보(in-vivo) 세포외 신경다발절(DRG) 기록법을 이용한 전기생리학적 약효평가법을 신약 개발 연구에 적용하여, 살아있는 동물에게 약물을 주입하고 전기생리학적으로 약물의 효과를 실시간으로 평가할 수 있도록 인비보(in-vivo) 세포외 신경다발절(DRG) 기록법을 이용한 전기생리학적 약효평가법을 제공하는 것을 기술적 과제로 한다.The purpose of the present invention is to apply an electrophysiological drug efficacy evaluation method using in-vivo extracellular nerve ganglion (DRG) recording to research on new drug development, inject a drug into a living animal, and electrophysiologically determine the effect of the drug. The technical task is to provide an electrophysiological drug efficacy evaluation method using in-vivo extracellular nerve ganglion (DRG) recording to enable real-time evaluation.
상기 과제를 해결하기 위하여, 본 발명에서는 하기와 같은 수단을 개시한다.In order to solve the above problems, the present invention discloses the following means.
일 양태에서, 본 발명은 인비보(in-vivo) 세포외(extracellular) 신경다발절(DRG) 기록법을 이용하여 전압 의존성 소디움 채널 억제제가 소디움 채널의 활성을 선택적으로 억제함으로써 세포막 전위가 활동전위를 유발하기 위한 역치(threshold)에 도달하는 것을 억제하거나 혹은 지연시켜 활동전위(AP)의 발생 빈도를 줄여주는 기전의 약물에 대한 약효를 평가하는 전기생리학적 약효 평가법에 있어서, (S1) 비히클(vehicle)을 투여하여 대조군 발화율(control firing rate) 구간의 활동전위 표준화된 발화율(normalized firing rate, %)를 측정하는 단계; (S2) 전압 의존성 소디움 채널 억제제를 투여하여 약물 발화율(firing rate) 구간의 활동전위 표준화된 발화율(normalized firing rate, %)를 측정하는 단계; 및 (S3) (S1)에서 측정된 활동전위 표준화된 발화율 대비 (S2)에서 측정된 활동전위 표준화된 발화율의 억제 정도를 통계적으로 확인하는 단계를 포함하고, 상기 (S1) 및 (S2)에서 활동전위 표준화된 발화율(normalized firing rate, %)은 하기 일반식 1을 통해 측정하는 것을 특징으로 하는 약효 평가법을 제공한다.In one aspect, the present invention uses in-vivo extracellular nerve ganglion (DRG) recording to use a voltage-dependent sodium channel inhibitor to selectively inhibit the activity of sodium channels, thereby causing the cell membrane potential to generate an action potential. In the electrophysiological drug efficacy evaluation method that evaluates the efficacy of drugs with a mechanism that reduces the frequency of action potential (AP) by inhibiting or delaying reaching the threshold for triggering, (S1) vehicle ) to measure the normalized firing rate (%) of action potentials in the control firing rate section; (S2) measuring the normalized firing rate (%) of action potentials in the drug firing rate section by administering a voltage-dependent sodium channel inhibitor; and (S3) statistically confirming the degree of inhibition of the action potential normalized firing rate measured in (S2) compared to the action potential normalized firing rate measured in (S1), and the activity in (S1) and (S2). Potential normalized firing rate (%) provides a drug efficacy evaluation method characterized in that it is measured using the general formula 1 below.
[일반식 1][General Formula 1]
Figure PCTKR2023012835-appb-img-000001
Figure PCTKR2023012835-appb-img-000001
상기 일반식 1에서, X는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 후 활동전위 표준화된 발화율(normalized firing rate, %)이고, A는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 전 각각의 von frey 그램 자극에 반응하여 발생되는 활동전위 발생 빈도(Hz)이며, B는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 직후인 전압 의존성 소디움 채널 억제제 발화율(firing rate) 구간의 각각의 von frey 그램 자극에 반응하여 발생되는 활동전위 발생 빈도(Hz)이다.In the above general formula 1, B is the action potential generation frequency (Hz) generated in response to von Frey gram stimulation, and B is each von Frey gram in the voltage-dependent sodium channel inhibitor firing rate section immediately after administration of vehicle or voltage-dependent sodium channel inhibitor. This is the frequency (Hz) of action potentials generated in response to stimulation.
Figure PCTKR2023012835-appb-img-000002
,
Figure PCTKR2023012835-appb-img-000003
Figure PCTKR2023012835-appb-img-000002
,
Figure PCTKR2023012835-appb-img-000003
또 다른 양태에서, 본 발명은 인비보(in-vivo) 세포외(extracellular) 신경다발절(DRG) 기록법을 이용하여 전압 의존성 소디움 채널 억제제가 소디움 채널의 활성을 선택적으로 억제함으로써 세포막 전위가 활동전위를 유발하기 위한 역치(threshold)에 도달하는 것을 억제하거나 혹은 지연시켜 활동전위의 발생 빈도를 줄여주는 기전의 약물에 대한 약효를 평가하는 전기생리학적 약효 평가법에 있어서, (S1') 비히클(vehicle)을 투여하여 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)을 측정하는 단계; (S2') 전압 의존성 소디움 채널 억제제를 투여하여 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)을 측정하는 단계; 및 (S3') (S1')에서 측정된 표준화된 활동전위의 역치값의 변화율 대비 (S2')에서 측정된 표준화된 활동전위의 역치값의 변화율의 증가 정도를 통계적으로 확인하는 단계를 포함하고, 상기 (S1') 및 (S2')에서 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)은 하기 일반식 2를 통해 측정하는 것을 특징으로 하는 약효 평가법을 제공한다.In another aspect, the present invention uses in-vivo extracellular nerve ganglion (DRG) recording to use a voltage-dependent sodium channel inhibitor to selectively inhibit the activity of sodium channels, thereby causing the cell membrane potential to become an action potential. In the electrophysiological drug efficacy evaluation method that evaluates the efficacy of drugs with a mechanism that reduces the frequency of action potentials by inhibiting or delaying reaching the threshold for inducing, (S1') vehicle Measuring the rate of change (normalized action potential threshold, %) of the threshold value of the normalized action potential by administering; (S2') measuring the normalized action potential threshold change rate (%) by administering a voltage-dependent sodium channel inhibitor; and (S3') statistically confirming the degree of increase in the rate of change in the threshold value of the standardized action potential measured in (S2') compared to the rate of change in the threshold value of the standardized action potential measured in (S1'). , the rate of change (normalized action potential threshold, %) of the normalized action potential threshold in (S1') and (S2') provides a drug efficacy evaluation method characterized in that it is measured using the following general formula 2.
[일반식 2][General Formula 2]
Figure PCTKR2023012835-appb-img-000004
Figure PCTKR2023012835-appb-img-000004
상기 일반식 2에서, Y는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 후 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)이고, C는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 전 활동전위를 발생시키는 최소의 von frey 그램 수이며, D는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 후 활동전위를 발생시키는 최소의 von frey 그램 수이다.In General Formula 2, Y is the normalized action potential threshold change rate (%) after administration of vehicle or voltage-dependent sodium channel inhibitor, and C is vehicle or voltage-dependent sodium channel D is the minimum number of von Frey grams that generate an action potential before administration of the inhibitor, and D is the minimum number of von Frey grams that generate an action potential after administration of a vehicle or voltage-dependent sodium channel inhibitor.
본 발명에 따른 인비보(in-vivo) 세포외 신경다발절(DRG) 기록법을 이용한 전기생리학적 약효평가법은 신약 개발을 위한 인비보 (in-vivo) 전기생리학적 약효 평가에 그대로 적용될 수 있는 이점이 있다.The electrophysiological drug efficacy evaluation method using in-vivo extracellular nerve ganglion (DRG) recording according to the present invention has the advantage of being directly applicable to in-vivo electrophysiological drug efficacy evaluation for new drug development. There is.
본 발명의 효과는 이상에서 언급한 효과들로 제한되지 않으며, 이하에서 설명할 내용으로부터 통상의 기술자에게 자명한 범위 내에서 다양한 효과들이 포함될 수 있다.The effects of the present invention are not limited to the effects mentioned above, and various effects may be included within the scope apparent to those skilled in the art from the contents described below.
도 1은 경구투여를 위한 삽관술 진행 과정을 나타낸 것이다.Figure 1 shows the intubation process for oral administration.
도 2는 본 발명에 따른 인비보 세포외 신경다발절(DRG) 기록법 (in vivo extracellular DRG recording method)의 모식도를 나타낸 것이다.Figure 2 shows a schematic diagram of the in vivo extracellular DRG recording method according to the present invention.
도 3은 본 발명에 따른 인비보 세포외 신경다발절(DRG) 기록법 (in vivo extracellular DRG recording method)을 이용한 전기생리학적 약효평가 실시예를 설명한 것이다.Figure 3 illustrates an example of electrophysiological drug efficacy evaluation using the in vivo extracellular DRG recording method according to the present invention.
도 4는 본 발명에 따른 인비보 세포외 신경다발절(DRG) 기록법 (in vivo extracellular DRG recording method)을 이용한 전기생리학적 약효평가 결과를 나타낸 것이다.Figure 4 shows the results of electrophysiological drug efficacy evaluation using the in vivo extracellular DRG recording method according to the present invention.
도 5는 본 발명에 따른 인비보 세포외 신경다발절(DRG) 기록법 (in vivo extracellular DRG recording method)을 이용한 전기생리학적 약효평가 결과를 나타낸 것이다.Figure 5 shows the results of electrophysiological drug efficacy evaluation using the in vivo extracellular DRG recording method according to the present invention.
이하, 본 명세서에 대하여 더욱 상세히 설명한다.Hereinafter, this specification will be described in more detail.
이를 구체적으로 설명하면 다음과 같다. 본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. 또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.This is explained in detail as follows. The terms used in this specification are general terms that are currently widely used as much as possible while considering the function in the present invention, but this may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless clearly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.
수치 범위는 상기 범위에 정의된 수치를 포함한다. 본 명세서에 걸쳐 주어진 모든 최대의 수치 제한은 낮은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 낮은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 최소의 수치 제한은 더 높은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 높은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 수치 제한은 더 좁은 수치 제한이 명확히 쓰여져 있는 것처럼, 더 넓은 수치 범위 내의 더 좋은 모든 수치 범위를 포함할 것이다.The numerical range includes the values defined in the range above. Every maximum numerical limit given throughout this specification includes all lower numerical limits as if the lower numerical limit were explicitly written out. Every minimum numerical limit given throughout this specification includes every higher numerical limit as if such higher numerical limit was clearly written. All numerical limits given throughout this specification will include all better numerical ranges within the broader numerical range, as if the narrower numerical limits were clearly written.
이하, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각에 대한 다른 설명 및 실시형태에도 적용될 수 있다. 즉, 본 발명에 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기에 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.Hereinafter, each description and embodiment disclosed in the present invention may also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description set forth below.
본 명세서에서 사용되는 「포함하는」과 같은 표현은, 다른 실시예를 포함할 가능성을 내포하는 개방형 용어 (open-ended terms)로 이해되어야 한다.Expressions such as “comprising” used in this specification should be understood as open-ended terms that imply the possibility of including other embodiments.
본 발명의 발명자들은 상기와 같은 과제를 해결하기 위해 연구 개발하던 중, 종래 이온채널의 변이에 의해 나타나는 전기생리학적인 특성을 인비보(in-vivo) 수준에서 확인 한 것이기에 약물의 투여 방법, 약물의 효과를 판단하기 위한 기준, 그리고 약효 평가를 위한 세부 절차와 핵심 요소 등은 고려되어 있지 않아서 신약 개발을 위한 인비보(in-vivo) 전기생리학적 약효 평가에는 그대로 적용될 수 없는 문제를 해결하기 위해 연구 개발 하였고, 인비보(in-vivo) 세포외 신경다발절 기록법을 이용할 경우 전압 의존성 소디움 채널 억제제의 임상시험에서의 예측 가능성을 높일 수 있고, 생체를 통해 임상에 상당히 유사한 환경에서 채널 레벨에서의 약효를 평가할 수 있음을 확인하여 본 발명을 완성하게 되었다.While conducting research and development to solve the above-mentioned problems, the inventors of the present invention confirmed the electrophysiological characteristics caused by mutations in conventional ion channels at the in-vivo level, thereby determining the method of drug administration and the use of the drug. Research was conducted to solve the problem that criteria for determining effectiveness and detailed procedures and key elements for drug efficacy evaluation were not taken into consideration and therefore could not be directly applied to in-vivo electrophysiological drug efficacy evaluation for new drug development. was developed, and the use of in-vivo extracellular nerve bundle recording method can increase the predictability in clinical trials of voltage-dependent sodium channel inhibitors, and drug efficacy at the channel level can be determined in an environment that is quite similar to clinical practice through in vivo studies. The present invention was completed by confirming that it can be evaluated.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
인비보(In vivo( in-vivoin-vivo ) 세포외 신경다발절(DRG) 기록법을 이용한 전기생리학적 약효평가법) Electrophysiological drug efficacy evaluation method using extracellular nerve ganglion (DRG) recording method
상기 과제를 해결하기 위하여, 본 발명에서는 하기와 같은 수단을 개시한다.In order to solve the above problems, the present invention discloses the following means.
일 양태에서, 본 발명은 인비보(in-vivo) 세포외(extracellular) 신경다발절(DRG) 기록법을 이용하여 전압 의존성 소디움 채널 억제제가 소디움 채널의 활성을 선택적으로 억제함으로써 세포막 전위가 활동전위를 유발하기 위한 역치(threshold)에 도달하는 것을 억제하거나 혹은 지연시켜 활동전위(AP)의 발생 빈도를 줄여주는 기전의 약물에 대한 약효를 평가하는 전기생리학적 약효 평가법에 있어서, (S1) 비히클(vehicle)을 투여하여 대조군 발화율(control firing rate) 구간의 활동전위 표준화된 발화율(normalized firing rate, %)를 측정하는 단계; (S2) 전압 의존성 소디움 채널 억제제를 투여하여 약물 발화율(firing rate) 구간의 활동전위 표준화된 발화율(normalized firing rate, %)를 측정하는 단계; 및 (S3) (S1)에서 측정된 활동전위 표준화된 발화율 대비 (S2)에서 측정된 활동전위 표준화된 발화율의 억제 정도를 통계적으로 확인하는 단계를 포함하고, 상기 (S1) 및 (S2)에서 활동전위 표준화된 발화율(normalized firing rate, %)은 하기 일반식 1을 통해 측정하는 것을 특징으로 하는 약효 평가법을 제공한다.In one aspect, the present invention uses in-vivo extracellular nerve ganglion (DRG) recording, wherein a voltage-dependent sodium channel inhibitor selectively inhibits the activity of the sodium channel, thereby causing the cell membrane potential to generate an action potential. In the electrophysiological drug efficacy evaluation method that evaluates the efficacy of drugs with a mechanism that reduces the frequency of action potential (AP) by suppressing or delaying reaching the threshold for triggering, (S1) vehicle ) to measure the normalized firing rate (%) of action potentials in the control firing rate section; (S2) measuring the normalized firing rate (%) of action potentials in the drug firing rate section by administering a voltage-dependent sodium channel inhibitor; and (S3) statistically confirming the degree of suppression of the action potential normalized firing rate measured in (S2) compared to the action potential normalized firing rate measured in (S1), and the activity in (S1) and (S2). Potential normalized firing rate (%) provides a drug efficacy evaluation method characterized in that it is measured using the general formula 1 below.
[일반식 1][General Formula 1]
Figure PCTKR2023012835-appb-img-000005
Figure PCTKR2023012835-appb-img-000005
상기 일반식 1에서, X는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 후 활동전위 표준화된 발화율(normalized firing rate, %)이고, A는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 전 각각의 von frey 그램 자극에 반응하여 발생되는 활동전위 발생 빈도(Hz)이며, B는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 직후인 전압 의존성 소디움 채널 억제제 발화율(firing rate) 구간의 각각의 von frey 그램 자극에 반응하여 발생되는 활동전위 발생 빈도(Hz)이다.In the above general formula 1, B is the action potential generation frequency (Hz) generated in response to von Frey gram stimulation, and B is each von Frey gram in the voltage-dependent sodium channel inhibitor firing rate section immediately after administration of vehicle or voltage-dependent sodium channel inhibitor. This is the frequency (Hz) of action potentials generated in response to stimulation.
Figure PCTKR2023012835-appb-img-000006
,
Figure PCTKR2023012835-appb-img-000007
.
Figure PCTKR2023012835-appb-img-000006
,
Figure PCTKR2023012835-appb-img-000007
.
본 발명에서 사용하는 용어는 다음과 같다.The terms used in the present invention are as follows.
본 발명에 있어서, 용어 「인비보(in-vivo) 세포외 신경다발절 기록법」이란, 마취된 상태의 마우스를 대상으로 신경다발절 뉴런 및 신경다발절 필라멘트 영역에서 발생되는 활동전위를 미세유리 파이펫을 이용하여 세포 외부에서 기록하는 방법을 의미한다.In the present invention, the term " in-vivo extracellular nerve bundle recording method" refers to the action potentials generated in nerve bundle neurons and nerve bundle filament areas in anesthetized mice using microglass pi. This refers to a method of recording from outside the cell using a pet.
본 발명에 있어서, 용어 「전압 의존성 소디움 채널 억제제」란, 특정 범위의 막전압에서 소디움 이온의 이동 통로가 되는 채널의 활성을 억제하는 저분자 화합물을 의미한다.In the present invention, the term “voltage-dependent sodium channel inhibitor” refers to a low-molecular-weight compound that inhibits the activity of a channel that serves as a passage for the movement of sodium ions in a specific range of membrane voltage.
본 발명에 있어서, 용어 「발생 빈도」란, 활동전위(action potential) 발생에 대한 빈번함의 정도를 의미한다.In the present invention, the term “frequency of occurrence” refers to the degree of frequency of occurrence of action potentials.
본 발명에 있어서, 용어 「대조군 발화율(control firing rate)」이란, 비히클(vehicle)을 투여한 후 각각 30분, 60분, 90분, 120분째에 폰 프레이(von frey) 자극을 통해 발생되는 2초 동안의 활동전위 발생 구간으로 비히클(vehicle) 투여 직전 시점과 비교하여 차이값을 도출하기위한 비히클(vehicle) 효과 구간을 의미한다.In the present invention, the term “control firing rate” refers to the 2 firing rate generated through von Frey stimulation at 30, 60, 90, and 120 minutes after administration of the vehicle, respectively. This is the action potential generation section for seconds and refers to the vehicle effect section for deriving the difference value compared to the time immediately before vehicle administration.
본 발명에 있어서, 용어 「약물 발화율(firing rate) 구간」이란, 약물을 투여한 후 각각 30분, 60분, 90분, 120분째에 폰 프레이(von frey) 자극을 통해 발생되는 2초 동안의 활동전위 발생 구간으로 약물 투여 직전 시점과 비교하여 차이값을 도출하기위한 약효 구간을 의미한다.In the present invention, the term “drug firing rate section” refers to the 2-second period generated through von Frey stimulation at 30, 60, 90, and 120 minutes after drug administration, respectively. This is the action potential generation section and refers to the drug effect section for deriving the difference value compared to the time immediately before drug administration.
본 발명에 있어서, 용어 「표준화된 발화율(normalized firing rate, %)」이란, von frey 자극에 반응하여 약물을 투여하기 전을 기준으로 약물을 투여 후 발생되는 활동전위 발화율의 변화 값을 백분율로 환산한 것을 의미한다.In the present invention, the term “normalized firing rate (%)” refers to the change in action potential firing rate that occurs after administering the drug compared to before administering the drug in response to von Frey stimulation, converted into a percentage. It means one thing.
본 발명에 있어서, 상기 약효 평가법은 하기 조건을 만족하는 것일 수 있다.In the present invention, the drug efficacy evaluation method may satisfy the following conditions.
조건 1: 상기 일반식 1에서 전압 의존성 소디움 체널 억제제를 투여한 경우, B 값은 A 값보다 작음 (B 값 < A 값)Condition 1: When the voltage-dependent sodium channel inhibitor is administered in General Formula 1 above, the B value is less than the A value (B value < A value)
조건 2: 상기 일반식 1에서, (S2)에서 측정된 활동전위 표준화된 발화율은 (S1)에서 측정된 활동전위 표준화된 발화율보다 작으며, 그 차이가 통계적으로 유의미한 경우임. Condition 2: In General Formula 1 above, the action potential normalized firing rate measured in (S2) is smaller than the action potential normalized firing rate measured in (S1), and the difference is statistically significant.
구체적으로, 통계적으로 유의미한 경우란, 아래에 설정한 통계적 검증을 통해 규정된 유의수준을 통과한 경우를 의미한다. Specifically, a statistically significant case means a case that passes the significance level specified through the statistical test set below.
통계의 귀무가설(null hypothesis)은 "(S2)에서 측정된 활동전위 표준화된 발화율은 (S1)에서 측정된 활동전위 표준화된 발화율보다 작지 않다." 이며, 대립가설(alternative hypothesis)은 "(S2)에서 측정된 활동전위 표준화된 발화율은 (S1)에서 측정된 활동전위 표준화된 발화율보다 작다." 이다. 유의수준은 보편적 기준인 p-값(p-value)<0.05로, 상기 조건을 만족한 경우 귀무가설을 기각하여 유의수준을 통과한 것으로 규정하였으며, 일반적으로 설정하는 P<0.05, P<0.01, P<0.001 3단계로 나누어 유의수준의 정도를 구분하였다. 실험 결과들을 mean ± SEM으로 나타내고, 대조군(S1)과 실험군(S2)에서 얻어진 결과 값들은 이원배치 분산분석(Two-way ANOVA)을 이용하여 사후 검정(post-hoc)을 거쳐 쌍별 비교(pairwise comparison)를 프리즘에서 제공하는 통계분석프로그램을 이용하여 시행하였다.The null hypothesis of statistics is "the action potential normalized firing rate measured in (S2) is not smaller than the action potential normalized firing rate measured in (S1)." , and the alternative hypothesis is “The action potential normalized firing rate measured in (S2) is smaller than the action potential normalized firing rate measured in (S1).” am. The significance level is the universal standard p-value <0.05. If the above conditions are met, the null hypothesis is rejected and the significance level is passed. The generally set values are P<0.05, P<0.01, P<0.001 The level of significance was divided into three levels. The experimental results are expressed as mean ± SEM, and the results obtained from the control group (S1) and the experimental group (S2) were subjected to a post-hoc pairwise comparison using two-way ANOVA. ) was performed using the statistical analysis program provided by Prism.
상기 조건 1과 조건 2를 만족함으로써, 비히클(vehicle) 대비 활동전위 표준화된 발화율을 억제하여 이온채널의 활성을 변화시킴으로써 통증의 억제 효과가 있음을 확인할 수 있다.By satisfying Condition 1 and Condition 2 above, it can be confirmed that there is a pain suppressing effect by changing the activity of ion channels by suppressing the action potential normalized firing rate compared to the vehicle.
본 발명에 있어서, 상기 전압 의존성 소디움 채널 억제제는 하기 화학식 1로 표시되는 화합물일 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the voltage-dependent sodium channel inhibitor may be a compound represented by the following formula (1), but is not limited thereto.
[화학식 1][Formula 1]
Figure PCTKR2023012835-appb-img-000008
Figure PCTKR2023012835-appb-img-000008
본 발명에 있어서, 상기 화학식 1로 표시되는 화합물은 Nav1.7 소디움 채널의 활성을 선택적으로 억제할 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the compound represented by Formula 1 can selectively inhibit the activity of the Nav1.7 sodium channel, but is not limited thereto.
본 발명에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 1 mg/kg 내지 100 mg/kg를 투여할 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, can be administered in an amount of 1 mg/kg to 100 mg/kg, but is not limited thereto.
본 발명에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 30 mg/kg를 투여할 수 있으며, 이에 한정되는 것은 아니다.In the present invention, 30 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, can be administered, but is not limited thereto.
본 발명에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 30 mg/kg을 투여할 때 상기 일반식 1에서, X, A 및 B는 하기와 같을 수 있으며, 이에 한정되는 것은 아니다.In the present invention, when 30 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, is administered, in Formula 1, X, A, and B may be as follows, but are not limited thereto. .
X: 0 % 내지 99.36306 %X: 0% to 99.36306%
A: 1.583333 내지 61.08333 HzA: 1.583333 to 61.08333 Hz
B: 0 내지 49.83333 HzB: 0 to 49.83333 Hz
본 발명에 있어서, 약효 평가법은, (S1) 단계 이전에 설치류 마취 후 척수(spinal cord)의 특정 타겟 부위에서 세포외 기록법을 수행하기 위해 척수 클램퍼(spinal cord clamper)를 이용하여 척추뼈를 고정하는 단계를 포함할 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the drug efficacy evaluation method involves fixing the vertebrae using a spinal cord clamper to perform extracellular recording in a specific target area of the spinal cord after anesthetizing a rodent before step (S1). It may include steps, but is not limited thereto.
본 발명에 있어서, 용어 「설치류」란, 마우스 (mouse) 또는 랫드 (rat)와 관련된 것을 의미한다.In the present invention, the term “rodent” means related to mouse or rat.
본 발명에 있어서, 상기 von frey 그램은 1 내지 100 g일 수 있고, 구체적으로는 1 g, 2 g, 4 g, 10 g 및 100 g 으로 이루어진 군에서 선택된 어느 하나 이상일 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the von Frey gram may be 1 to 100 g, specifically, may be any one or more selected from the group consisting of 1 g, 2 g, 4 g, 10 g, and 100 g, and is limited thereto. no.
또 다른 일 양태로서, 본 발명은 인비보(in-vivo) 세포외(extracellular) 신경다발절(DRG) 기록법을 이용하여 전압 의존성 소디움 채널 억제제가 소디움 채널의 활성을 선택적으로 억제함으로써 세포막 전위가 활동전위를 유발하기 위한 역치(threshold)에 도달하는 것을 억제하거나 혹은 지연시켜 활동전위의 발생 빈도를 줄여주는 기전의 약물에 대한 약효를 평가하는 전기생리학적 약효 평가법에 있어서, (S1') 비히클(vehicle)을 투여하여 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)을 측정하는 단계; (S2') 전압 의존성 소디움 채널 억제제를 투여하여 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)을 측정하는 단계; 및 (S3') (S1')에서 측정된 표준화된 활동전위의 역치값의 변화율 대비 (S2')에서 측정된 표준화된 활동전위의 역치값의 변화율의 증가 정도를 통계적으로 확인하는 단계를 포함하고, 상기 (S1') 및 (S2')에서 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)은 하기 일반식 2를 통해 측정하는 것을 특징으로 하는 약효 평가법을 제공한다.In another aspect, the present invention uses in-vivo extracellular nerve ganglion (DRG) recording to use a voltage-dependent sodium channel inhibitor to selectively inhibit the activity of sodium channels, thereby increasing the cell membrane potential. In an electrophysiological drug efficacy evaluation method that evaluates the efficacy of drugs with a mechanism that reduces the frequency of action potentials by inhibiting or delaying reaching the threshold for inducing potentials, (S1') vehicle ) to measure the rate of change (normalized action potential threshold, %) of the threshold value of the normalized action potential; (S2') measuring the normalized action potential threshold change rate (%) by administering a voltage-dependent sodium channel inhibitor; and (S3') statistically confirming the degree of increase in the rate of change in the threshold value of the standardized action potential measured in (S2') compared to the rate of change in the threshold value of the standardized action potential measured in (S1'). , the rate of change (normalized action potential threshold, %) of the normalized action potential threshold in (S1') and (S2') provides a drug efficacy evaluation method characterized in that it is measured using the following general formula 2.
[일반식 2][General Formula 2]
Figure PCTKR2023012835-appb-img-000009
Figure PCTKR2023012835-appb-img-000009
상기 일반식 2에서, Y는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 후 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)이고, C는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 전 활동전위를 발생시키는 최소의 von frey 그램 수이며, D는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 후 활동전위를 발생시키는 최소의 von frey 그램 수이다.In General Formula 2, Y is the normalized action potential threshold change rate (%) after administration of vehicle or voltage-dependent sodium channel inhibitor, and C is vehicle or voltage-dependent sodium channel D is the minimum number of von Frey grams that generate an action potential before administration of the inhibitor, and D is the minimum number of von Frey grams that generate an action potential after administration of a vehicle or voltage-dependent sodium channel inhibitor.
본 발명에 있어서, 용어 「표준화된 활동전위의 역치값의 변화율 (normalized action potential threshold, %)」이란, 약물을 투여하기 전을 기준으로 약물을 투여 후 활동전위를 유발하기 위해 필요한 최소의 자극의 변화값을 백분율로 환산한 것을 의미한다.In the present invention, the term “normalized action potential threshold (%)” refers to the minimum stimulation required to induce an action potential after administration of the drug, based on before administration of the drug. This means that the change value has been converted into a percentage.
본 발명에 있어서, 상기 약효 평가법은 하기 조건을 만족하는 것일 수 있다.In the present invention, the drug efficacy evaluation method may satisfy the following conditions.
조건 3: 상기 일반식 2에서, 전압 의존성 소디움 체널 억제제를 투여한 경우, D 값은 C 값보다 큼 (D 값 > C 값)Condition 3: In the general formula 2 above, when a voltage-dependent sodium channel inhibitor is administered, the D value is greater than the C value (D value > C value)
조건 4: 상기 일반식 2에서, (S2')에서 측정된 표준화된 활동전위의 역치값의 변화율은 (S1')에서 측정된 표준화된 활동전위의 역치값의 변화율보다 크며, 그 차이가 통계적으로 유의미한 경우임.Condition 4: In the above general formula 2, the rate of change of the threshold value of the standardized action potential measured in (S2') is greater than the rate of change of the threshold value of the standardized action potential measured in (S1'), and the difference is statistically This is a significant case.
구체적으로, 통계적으로 유의미한 경우란, 아래에 설정한 통계적 검증을 통해 규정된 유의수준을 통과한 경우를 의미한다. Specifically, a statistically significant case means a case that passes the significance level specified through the statistical test set below.
통계의 귀무가설(null hypothesis)은 "(S2')에서 측정된 표준화된 활동전위의 역치값의 변화율은 (S1')에서 측정된 표준화된 활동전위의 역치값의 변화율보다 크지 않다." 이며, 대립가설(alternative hypothesis)은 "(S2')에서 측정된 표준화된 활동전위의 역치값의 변화율은 (S1')에서 측정된 표준화된 활동전위의 역치값의 변화율보다 크다." 이다. 유의수준은 보편적 기준인 p-값(p-value)<0.05로, 상기 조건을 만족한 경우 귀무가설을 기각하여 유의수준을 통과한 것으로 규정하였으며, 일반적으로 설정하는 P<0.05, P<0.01, P<0.001 3단계로 나누어 유의수준의 정도를 구분하였다. 실험 결과들을 mean ± SEM으로 나타내고, 대조군(S1')과 실험군(S2')에서 얻어진 결과 값들은 이원배치 분산분석(Two-way ANOVA)을 이용하여 사후 검정(post-hoc)을 거쳐 쌍별 비교(pairwise comparison)를 프리즘에서 제공하는 통계분석프로그램을 이용하여 시행하였다.The null hypothesis of statistics is "The rate of change in the threshold value of the standardized action potential measured in (S2') is not greater than the rate of change in the threshold value of the standardized action potential measured in (S1')." , and the alternative hypothesis is "The rate of change in the threshold value of the standardized action potential measured in (S2') is greater than the rate of change in the threshold value of the standardized action potential measured in (S1')." am. The significance level is the universal standard p-value <0.05. If the above conditions are met, the null hypothesis is rejected and the significance level is passed. The generally set values are P<0.05, P<0.01, P<0.001 The level of significance was divided into three levels. The experimental results are expressed as mean ± SEM, and the results obtained from the control group (S1') and the experimental group (S2') were compared pairwise through a post-hoc test using two-way ANOVA ( Pairwise comparison) was performed using the statistical analysis program provided by Prism.
상기 조건 3과 조건 4를 만족함으로써, 비히클(vehicle) 대비 활동전위 표준화된 발화율을 억제함으로써 전압 의존성 소디움 채널 억제제의 투여로 인하여 활동전위(AP)를 발생시킬 수 있는 자극의 역치값을 높여줌으로써 통증의 억제 효과가 있음을 확인할 수 있다.By satisfying the conditions 3 and 4 above, pain is reduced by suppressing the normalized firing rate of action potentials compared to the vehicle, thereby increasing the threshold value of the stimulus that can generate action potentials (AP) due to the administration of a voltage-dependent sodium channel inhibitor. It can be confirmed that there is an inhibitory effect.
본 발명에 있어서, 상기 전압 의존성 소디움 채널 억제제는 하기 화학식 1로 표시되는 화합물일 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the voltage-dependent sodium channel inhibitor may be a compound represented by the following formula (1), but is not limited thereto.
[화학식 1][Formula 1]
Figure PCTKR2023012835-appb-img-000010
Figure PCTKR2023012835-appb-img-000010
본 발명에 있어서, 상기 화학식 1로 표시되는 화합물은 Nav1.7 소디움 채널의 활성을 선택적으로 억제할 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the compound represented by Formula 1 can selectively inhibit the activity of the Nav1.7 sodium channel, but is not limited thereto.
본 발명에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 1 mg/kg 내지 100 mg/kg를 투여할 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, can be administered in an amount of 1 mg/kg to 100 mg/kg, but is not limited thereto.
본 발명에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 30 mg/kg를 투여할 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, can be administered at 30 mg/kg, but is not limited thereto.
본 발명에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 30 mg/kg을 투여할 때 상기 일반식 2에서, Y, C 및 D는 하기와 같을 수 있으며, 이에 한정되는 것은 아니다.In the present invention, when administering 30 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, in Formula 2, Y, C and D may be as follows, but are not limited thereto. .
Y: 50 % 내지 1000 %Y: 50% to 1000%
C: 0.04 내지 4 gC: 0.04 to 4 g
D: 0.02 내지 10 g.D: 0.02 to 10 g.
본 발명에 있어서, 상기 약효 평가법은, (S1') 단계 이전에 설치류 마취 후 척수(spinal cord)의 특정 타겟 부위에서 세포외 기록법을 수행하기 위해 척수 클램퍼(spinal cord clamper)를 이용하여 척추뼈를 고정하는 단계를 포함할 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the drug efficacy evaluation method uses a spinal cord clamper to perform extracellular recording in a specific target area of the spinal cord after anesthetizing a rodent before the (S1') step. It may include a fixing step, but is not limited thereto.
본 발명에 있어서, 용어 「설치류」란, 마우스 (mouse) 또는 랫드 (rat)와 관련된 것을 의미한다. In the present invention, the term “rodent” means related to mouse or rat.
실시예Example
실시예 1: 실험동물(마우스)의 준비Example 1: Preparation of experimental animals (mice)
본 발명에 따른 인비보(in-vivo) 세포외 신경다발절(DRG) 기록법을 이용한 전기생리학적 약효평가를 위해서, 마우스를 사용하였다. 사용된 마우스의 품종은 ICR이고, 10 마리를 사용하였으며, 성별은 숫컷이고, 대상 연령은 5~7주였으며, 초기 체중은 25~40 g 이였고, ㈜코아텍에서 공급받아 사용하였다.For electrophysiological drug efficacy evaluation using the in-vivo extracellular nerve ganglion (DRG) recording method according to the present invention, mice were used. The breed of mice used was ICR, 10 mice were used, the gender was male, the target age was 5 to 7 weeks, the initial weight was 25 to 40 g, and they were supplied by Coretech Co., Ltd.
마취는 멸균된 1cc 주사기(26 gage)를 이용하여 우레탄(urethane)을 1.2 g/kg 용량으로 복강 주사하여 신경다발절(DRG) 노출을 위한경정맥 삽관을 위한 준비를 하였다.Anesthesia was administered by intraperitoneal injection of urethane at a dose of 1.2 g/kg using a sterilized 1cc syringe (26 gage) to prepare for jugular cannulation for exposure of the nerve bundle ganglion (DRG).
실시예 2: 경구투여를 위한 삽관Example 2: Intubation for oral administration
약물 투여 경로를 확보하기 위해서 경구 투여용 삽관술을 수행하였고, 도 2에 나타내었다. 도 2를 참조하면, 경구 투여용 삽관을 위한 의료용 폴리에틸렌 튜빙은 내경 0.011인치, 외경 0.024인치의 PE 10을 사용하였다. 마취된 마우스의 위(stomach)에 폴리에틸렌 튜빙(PE 10)을 위치시키기 위해서 경구 투여용 주사침(zonde for oral administration, 마우스용, 1.2 in X 1.9 mm, 20 G)를 가이드로 이용한 뒤 폴리에틸렌 튜빙(PE 10) 만을 남기고 경구 투여용 주사침을 다시 제거하였다.Intubation for oral administration was performed to secure the drug administration route, as shown in Figure 2. Referring to Figure 2, medical polyethylene tubing for intubation for oral administration was PE 10 with an inner diameter of 0.011 inches and an outer diameter of 0.024 inches. In order to place polyethylene tubing (PE 10) in the stomach of an anesthetized mouse, a needle for oral administration (zonde for oral administration, mouse, 1.2 in 10) The needle for oral administration was removed again, leaving only the remaining one.
실시예 3: 전기생리학적 기록을 위한 신경다발절(DRG) 노출Example 3: Exposure of nerve bundle ganglia (DRG) for electrophysiological recordings
인비보(in-vivo) 세포외 신경다발절(DRG) 기록법을 이용한 전기생리학적 기록을 위해서 마우스 추궁절제술(laminectomy)을 수행하였다. 추궁절제술을 위해서 마우스 등쪽 표피 중심부를 절개하여 척추뼈를 노출시키고, 척수 클램퍼(spinal cord clamper)를 이용하여 마우스의 척추뼈를 움직이지 않도록 단단히 고정시켰다. 이때 척추뼈는 L4와 L5 척추뼈 사이로 척수(spinal cord)가 보이도록 각도를 조절하여 고정하였고, 이 중심부를 기준으로 미니 드릴(mini-drill)을 사용하여 바깥쪽으로 척수(spinal cord)와 연결된 신경다발절(DRG)이 보이도록 노출시켰다. 마우스의 배쪽 부분에는 온열 패드(heating pad)를 깔아 차가운 바닥과 직접 닿지 않고 체온이 유지되도록 하였으며, 양쪽 눈은 마르지 않도록 안연고(eye ointment)를 충분히 도포해주었다. 노출된 척수(spinal cord)와 신경다발절(DRG) 조직이 마르는 것을 예방하고, 실험 진행 시 주변 조직으로부터 발생되는 전기적인 노이즈를 차단하기 위해서 마우스 등쪽 표피의 사방을 실로 묶어 수조를 만든 뒤 미네랄 오일(mineral oil)로 채워주었다.Mouse laminectomy was performed for electrophysiological recording using in-vivo extracellular nerve ganglion (DRG) recording. For laminectomy, an incision was made in the center of the epidermis on the back of the mouse to expose the vertebrae, and the vertebrae of the mouse were firmly fixed so as not to move using a spinal cord clamper. At this time, the vertebrae were fixed by adjusting the angle so that the spinal cord was visible between the L4 and L5 vertebrae, and the nerves connected to the spinal cord were moved outward using a mini-drill based on this center. The multiple nodes (DRG) were exposed to be visible. A heating pad was placed on the ventral side of the mouse to maintain body temperature without direct contact with the cold floor, and eye ointment was applied liberally to both eyes to prevent them from drying out. To prevent the exposed spinal cord and nerve bundle ganglion (DRG) tissue from drying out and to block electrical noise generated from surrounding tissues during the experiment, a water tank was created by tying the epidermis on the back of the mouse with thread on all sides and oiled with mineral oil. It was filled with (mineral oil).
실시예 4: 인비보(Example 4: In vivo ( in-vivoin-vivo ) 세포외 신경다발절(DRG) 기록) Extracellular ganglion (DRG) recordings.
인비보(in-vivo) 세포외 기록법(extracellular recording method)을 진행하기 위해, 직경 1 ~ 2 μm 정도의 미세한 유리 피펫을 사용하였다. 저항은 10~20 MΩ 정도 되는 유리 피펫을 풀러(puller)로 만들어 준비하였으며, 피펫 안에는 인공뇌척수액 (artificial cerebrospinal fluid; ACSF) 용액 (124 mM NaCl, 1.3 mM MgSO4, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 2.4 mM CaCl2, 및 10 mM glucose)을 채워서 기록에 사용하였다. 먼저 신경다발절(DRG)을 감싸고 있는 경막(dura)을 미세 유리 피펫을 이용하여 미세하게 찢어서 세포외 기록법(extracellular recording method)을 진행 시 피펫 끝의 손상을 예방하였고, 인공뇌척수액(artificial cerebrospinal fluid; ACSF)이 채워진 피펫을 신경다발절(DRG) 영역에 위치시킨 후, 미세조작기(micromanipulator)를 이용하여 수직으로 피펫을 내려서 신경다발절(DRG) 뉴런(neuron)으로부터 발생되는 활동전위(AP)를 기록하였다. 무작위로 많은 신경다발절(DRG) 뉴런(neuron) 중 하나에 피펫을 근접시킨 후 자극에 대한 반응이 있는지를 확인하기 위하여 마우스의 무릎 또는 발바닥을 붓으로 쓸면서 활동전위(AP) 발생을 관찰하였다. 자극에 대한 반응이 없는 경우, 피펫을 이동시켜서 다른 신경다발절(DRG) 뉴런(neuron)에 위치시키고, 다시 붓(Brush)으로 자극하여 활동전위(AP)가 발생되는 뉴런(neuron)을 찾아 실험을 수행하였다 (도 2 참조).To perform the in-vivo extracellular recording method, a fine glass pipette with a diameter of approximately 1 to 2 μm was used. The resistance was prepared by making a glass pipette with a resistance of about 10 to 20 MΩ using a puller, and inside the pipette was an artificial cerebrospinal fluid (ACSF) solution (124 mM NaCl, 1.3 mM MgSO 4 , 3 mM KCl, 1.25 mM NaH 2 PO 4 , 26mM NaHCO 3 , 2.4mM CaCl 2 , and 10mM glucose) were filled and used for recording. First, the dura surrounding the nerve bundle ganglion (DRG) was finely torn using a fine glass pipette to prevent damage to the tip of the pipette during the extracellular recording method, and artificial cerebrospinal fluid (artificial cerebrospinal fluid) was used. After placing the pipette filled with ACSF) in the nerve bundle ganglion (DRG) area, lower the pipette vertically using a micromanipulator to capture the action potential (AP) generated from the nerve bundle ganglion (DRG) neuron. It was recorded. After randomly placing a pipette close to one of many DRG neurons, action potential (AP) generation was observed by sweeping the knee or sole of the mouse with a brush to check whether there was a response to stimulation. . If there is no response to stimulation, move the pipette and place it on another nerve bundle ganglion (DRG) neuron, then stimulate it again with a brush to find the neuron that generates an action potential (AP) and experiment. was performed (see Figure 2).
실시예 5: 전기생리학적 약효 평가Example 5: Electrophysiological drug efficacy evaluation
약물의 효과를 평가하기 위해서 특정 그램(g)의 von frey 자극(1 g, 2 g, 4 g, 10 g, 100 g; 5초 간격으로 5초간 3번 자극)에 반응하여 신경다발절(DRG) 뉴런(neuron)에서 발생되는 활동전위(AP) 발화율의 변화값(발화율 테스트, firing rate test; FT)과 활동전위(AP) 유발에 필요한 최소 자극(von frey)의 크기에 대한 변화(역치 테스트, threshold test; TT)를 관찰하였다 (도 3 참조). To evaluate the effect of a drug, nerve bundle ganglia (DRG) were stimulated in response to specific grams (g) of von Frey stimulation (1 g, 2 g, 4 g, 10 g, 100 g; three stimulations for 5 seconds at 5-second intervals). ) Changes in the action potential (AP) firing rate generated from neurons (firing rate test; FT) and changes in the size of the minimum stimulus (von Frey) required to induce an action potential (AP) (threshold test) , threshold test (TT) was observed (see Figure 3).
평가 약물은 전압 의존성 소디움 채널 억제제인 하기 화학식 1로 표시되는 화합물 (iN1011-N17, 30 mg/kg; 경구투여)로서, 다양한 전압 의존성 소디움 채널 중 특정 아형(Nav1.7)의 소디움 채널의 활성을 선택적으로 억제함으로써, 세포막 전위가 활동전위(AP)를 유발하기 위한 역치(threshold)에 도달하는 것을 억제 혹은 지연시켜 활동전위(AP)의 발생 빈도를 줄여주는 기전의 약물을 사용하였다.The evaluation drug is a voltage-dependent sodium channel inhibitor, a compound represented by the following formula (iN1011-N17, 30 mg/kg; oral administration), which inhibits the activity of a specific subtype (Nav1.7) of sodium channels among various voltage-dependent sodium channels. A drug was used that reduces the frequency of action potential (AP) occurrence by selectively inhibiting or delaying the cell membrane potential from reaching the threshold for inducing an action potential (AP).
[화학식 1][Formula 1]
Figure PCTKR2023012835-appb-img-000011
Figure PCTKR2023012835-appb-img-000011
약효 평가를 위한 첫번째 파라미터는 발화율의 변화값(FT)으로서, von frey 자극에 의한 활동전위(AP)의 발생 빈도 (Hz)를 사용하였다. 한 개체에서 iN1011-N17 (화학식 1 화합물, 실험군) 혹은 vehicle(대조군, 증류수(distilled water)에 1% Tween® 80 (polysorbate)와 0.5% Methylcellulose를 첨가한 것 만을 처리, 이하 동일)을 경구 투여하기 전 각각의 von frey 그램(g) 자극에 반응하여 발생하는 활동전위(AP)의 발생 빈도(before firing rate) 대비, iN1011-N17 (화학식 1 화합물, 실험군) 혹은 vehicle(대조군)의 경구 투여 후 30분, 60분, 90분 및 120분 뒤 von frey 그램(g) 자극에 반응하여 발생하는 활동전위(AP)의 발생 빈도(after firing rate; 30분, 60분, 90분 및 120분)의 변화를 측정하여 약효를 평가하였다 (도 3 및 도 4 참조).The first parameter for drug efficacy evaluation was the change in firing rate (FT), which used the frequency (Hz) of action potentials (AP) generated by von Frey stimulation. Orally administering iN1011-N17 (formula 1 compound, experimental group) or vehicle (control group, treated only with 1% Tween® 80 (polysorbate) and 0.5% Methylcellulose added to distilled water, same hereinafter) to one subject. After oral administration of iN1011-N17 (formula 1 compound, experimental group) or vehicle (control group), compared to the firing rate of action potentials (AP) generated in response to each von Frey gram (g) stimulus, 30 Changes in the after firing rate (30, 60, 90, and 120 min) of action potentials (APs) in response to von Frey gram (g) stimulation at 30, 60, 90, and 120 min. The drug efficacy was evaluated by measuring (see Figures 3 and 4).
또한 약효 평가를 위한 두번째 파라미터는 최소 자극(von frey)의 크기에 대한 변화(TT)로서, 활동전위(AP)를 발생시키는 최소한의 von frey 그램(g) 자극을 사용하였다. 즉, 한 개체에서 iN1011-N17 (화학식 1 화합물, 실험군) 혹은 vehicle(대조군)을 경구 투여하기 전 활동전위(AP) 발생에 필요한 최소의 von frey 자극에 대한 그램(before threshold)을 측정하고, 투여 후 각각 30분, 60분, 90분 및 120분 뒤 동일하게 활동전위(AP) 발생에 필요한 최소의 von frey 자극 그램(after threshold)을 측정하여 약효를 평가하였다 (도 3 및 도 5 참조).In addition, the second parameter for drug efficacy evaluation is the change (TT) in the size of the minimum stimulus (von Frey), and the minimum von Frey gram (g) stimulus that generates an action potential (AP) was used. In other words, before oral administration of iN1011-N17 (Formula 1 compound, experimental group) or vehicle (control group) in one subject, the grams (before threshold) for the minimum von Frey stimulation required to generate an action potential (AP) are measured, and administered. The drug efficacy was evaluated by measuring the minimum von Frey stimulation gram (after threshold) required to generate an action potential (AP) at 30, 60, 90, and 120 minutes, respectively (see Figures 3 and 5).
[일반식 1][General Formula 1]
Figure PCTKR2023012835-appb-img-000012
Figure PCTKR2023012835-appb-img-000012
상기 일반식 1에서, A와 B는 하기와 같다.In General Formula 1, A and B are as follows.
Figure PCTKR2023012835-appb-img-000013
Figure PCTKR2023012835-appb-img-000013
Figure PCTKR2023012835-appb-img-000014
Figure PCTKR2023012835-appb-img-000014
상기 일반식 1에서 X는 도 4의 결과값을 도출하기 위한 활동전위(AP) 발화율(normalized firing rate, %)이고, A는 비히클(vehicle) 또는 약물 투여 전 구간내 각각의 von frey 그램 자극에(1 g, 2 g, 4 g, 10 g, 100 g) 반응하여 발생되는 활동전위(AP) 발생 빈도(Hz)를 나타내며, B는 비히클(vehicle) 또는 약물 투여 후 30, 60, 90, 120분 구간내에서 각각의 von frey 그램 자극에(1 g, 2 g, 4 g, 10 g, 100 g) 반응하여 발생되는 활동전위(AP) 빈도(Hz)이다. 활동전위(AP)가 1초당 1회 발생한 경우 1 Hz이기 때문에, 2초 동안 각각의 von frey 자극에 의해 발생되는 3구간의 활동전위(AP) 개수의 평균값을 2로 나누어 각각의 구간내 von frey 자극별로 활동전위(AP)의 발생 빈도를 구하였다. 위와 같이 도출된 A값과 B값을 이용하여 비히클(vehicle) 또는 약물 투여 후 활동전위(AP) 발화율(%)인 X값을 도출하였다.In the above general formula 1, (1 g, 2 g, 4 g, 10 g, 100 g) Represents the action potential (AP) generation frequency (Hz) generated in response, and B is 30, 60, 90, 120 after vehicle or drug administration. This is the action potential (AP) frequency (Hz) generated in response to each von Frey gram stimulus (1 g, 2 g, 4 g, 10 g, 100 g) within a minute interval. Since the rate is 1 Hz when an action potential (AP) occurs once per second, the average value of the number of action potentials (AP) in 3 sections generated by each von Frey stimulus for 2 seconds is divided by 2 to determine the von Frey rate within each section. The frequency of action potential (AP) occurrence was calculated for each stimulus. Using the A and B values derived as above, the X value, which is the action potential (AP) firing rate (%) after vehicle or drug administration, was derived.
구체적으로, 도 4와 표 1 내지 5를 참조하면, 전압 의존성 소디움 채널 억제제인 iN1011-N17의 투여군(화학식 1 화합물, 실험군)은 비히클(vehicle)을 투여한 군(대조군)과는 다르게 모든 개체에서 자극의 강도와는 무관하게 투여 전보다 약물 투여 후 더 낮은 신경다발절(DRG)에서의 활동전위(AP) 발생빈도가 기록되었다. 이는 대조군과 비교하여 명확하게 다른 양상으로 그 차이는 통계적으로도 유의미한 감소임이 확인되었다. 더하여, 이러한 실험군의 약물 효과는 약물 투여 후 2 시간까지 그 효과가 지속되었으며, 시간이 지남에 따라 활동전위(AP)의 발생빈도를 억제시키는 정도가 점차 강화되는 경향성을 확인할 수 있었다. 본 결과는 전압 의존성 소디움 채널 억제제인 iN1011-N17의 투여가 활동전위(AP)의 발생빈도를 억제시킴으로써 통증을 전달하는 신호를 효과적으로 억제시켜 수 시간까지 효과적인 진통 효과를 나타낼 수 있음을 의미한다. 따라서 본 발명은 이온채널의 활성을 변화시켜 통증을 억제하는 기전을 갖는 약물 개발과정에서 개발 약물작용의 개념 증명(proof of concept; POC) 및 매카니즘 증명(proof of mechanism; POM) 확보에 어려움을 극복할 수 있는 확실한 대안법으로 판단된다.Specifically, referring to Figure 4 and Tables 1 to 5, the group administered iN1011-N17, a voltage-dependent sodium channel inhibitor (formula 1 compound, experimental group), was different from the group administered the vehicle (control group) in all subjects. Regardless of the intensity of stimulation, a lower frequency of action potential (AP) occurrence in the nerve bundle ganglion (DRG) was recorded after drug administration than before administration. This was clearly different compared to the control group, and the difference was confirmed to be a statistically significant decrease. In addition, the drug effect in this experimental group lasted up to 2 hours after drug administration, and it was confirmed that the degree to which the frequency of action potential (AP) was suppressed gradually strengthened over time. These results mean that administration of iN1011-N17, a voltage-dependent sodium channel inhibitor, can effectively suppress signals that transmit pain by suppressing the frequency of action potentials (AP), producing an effective analgesic effect for up to several hours. Therefore, the present invention overcomes the difficulty in securing proof of concept (POC) and proof of mechanism (POM) of the developed drug action in the process of developing a drug with a mechanism to suppress pain by changing the activity of ion channels. It is judged to be a reliable alternative method.
von frey 자극
(1 g)von frey stimulation
(1 g) 		Vehicle (Hz)Vehicle (Hz) Normalized firing rate (%)Normalized firing rate (%) iN1011-N17 (Hz)iN1011-N17 (Hz) Normalized firing rate (%)Normalized firing rate (%)
BeforeBefore AfterAfter BeforeBefore AfterAfter
30 min30min 2.3333332.333333 2.3333332.333333 100100 2.6666672.666667 0.50.5 18.7518.75
33 4.54.5 150150 1.5833331.583333 0.3333330.333333 21.0526321.05263
4.254.25 4.3333334.333333 101.9608101.9608 2.0833332.083333 1One 4848
5.8333335.833333 5.6666675.666667 97.1428697.14286 7.8333337.833333 6.1666676.166667 78.723478.7234
16.1666716.16667 1818 111.3111.3
13.513.5 1414 103.7103.7
평균average 110.6839±8.1110.6839±8.1 41.632±14.041.632±14.0
60 min60min 2.3333332.333333 2.52.5 107.1429107.1429 2.6666672.666667 0.3333330.333333 12.512.5
33 4.1666674.166667 138.8889138.8889 1.5833331.583333 0.1666670.166667 10.5263210.52632
4.254.25 4.1666674.166667 98.0392298.03922 2.0833332.083333 1.666671.66667 8080
5.8333335.833333 5.55.5 94.2857194.28571 7.8333337.833333 5.55.5 70.2127770.21277
16.1666716.16667 20.8333320.83333 128.9128.9
13.513.5 10.6666710.66667 7979
평균average 107.7095±9.2107.7095±9.2 43.310±18.543.310±18.5
90 min90min 2.3333332.333333 2.52.5 107.1429107.1429 2.6666672.666667 00 00
33 2.8333332.833333 94.4444494.44444 1.5833331.583333 1One 63.1578963.15789
4.254.25 4.3333334.333333 101.9608101.9608 2.0833332.083333 1.51.5 7272
5.8333335.833333 6.56.5 111.4286111.4286 7.8333337.833333 77 89.361789.3617
16.1666716.16667 1717 105.2105.2
13.513.5 1313 96.396.3
평균average 102.7461±2.7102.7461±2.7 56.130±19.556.130±19.5
120 min120min 2.3333332.333333 33 128.5714128.5714 2.6666672.666667 0.1666670.166667 6.256.25
33 33 100100 1.5833331.583333 0.1666670.166667 10.5263210.52632
4.254.25 4.54.5 105.8824105.8824 2.0833332.083333 00 00
5.8333335.833333 77 120120 7.8333337.833333 2.6666672.666667 34.0425534.04255
16.1666716.16667 14.7514.75 91.291.2
13.513.5 11.3333311.33333 8484
평균average 104.9423±6.9104.9423±6.9 12.705±7.412.705±7.4
von frey 자극
(2 g)von frey stimulation
(2 g) 		Vehicle (Hz)Vehicle (Hz) Normalized firing rate (%)Normalized firing rate (%) iN1011-N17 (Hz)iN1011-N17 (Hz) Normalized firing rate (%)Normalized firing rate (%)
BeforeBefore AfterAfter BeforeBefore AfterAfter
30 min30min 4.1666674.166667 44 95.9999995.99999 8.4583338.458333 6.1666676.166667 72.9064172.90641
7.68757.6875 8.3333338.333333 108.4011108.4011 7.2777787.277778 3.6666673.666667 50.3816850.38168
5.0833335.083333 5.255.25 103.2787103.2787 2.6666672.666667 0.6666670.666667 25.0000125.00001
13.1666713.16667 1515 113.924113.924 35.4166735.41667 16.3333316.33333 46.1176346.11763
41.2541.25 38.1666738.16667 92.5252692.52526
17.8333317.83333 18.6666718.66667 104.6729104.6729
평균average 103.134±3.2103.134±3.2 48.601±9.848.601±9.8
60 min60min 4.1666674.166667 3.53.5 83.9999983.99999 8.6666678.666667 5.3333335.333333 61.5384661.53846
7.68757.6875 8.58.5 110.5691110.5691 7.2777787.277778 0.8333330.833333 11.4503811.45038
5.0833335.083333 55 98.3606698.36066 2.6666672.666667 0.50.5 18.7518.75
13.1666713.16667 11.1666711.16667 84.8101384.81013 35.4166735.41667 29.529.5 83.2941183.29411
41.2541.25 36.6666736.66667 88.888988.8889
17.8333317.83333 16.516.5 92.5233892.52338
평균average 93.192±4.193.192±4.1 43.758±17.243.758±17.2
90 min90min 4.1666674.166667 4.6666674.666667 112112 8.4583338.458333 6.8333336.833333 80.7881880.78818
7.68757.6875 1010 130.0813130.0813 7.2777787.277778 1.6666671.666667 22.9007722.90077
5.0833335.083333 55 98.3606698.36066 2.6666672.666667 1.6666671.666667 62.562.5
13.1666713.16667 1313 98.7341598.73415 35.4166735.41667 18.8333318.83333 53.1764653.17646
41.2541.25 39.8333339.83333 96.5656596.56565
17.8333317.83333 1515 84.1121784.11217
평균average 103.309±6.5103.309±6.5 54.841±12.154.841±12.1
120 min120min 4.1666674.166667 3.53.5 83.9999983.99999 8.6666678.666667 77 80.7692380.76923
7.68757.6875 77 91.0569191.05691 7.2777787.277778 1.1666671.166667 16.0305416.03054
5.0833335.083333 5.55.5 108.1967108.1967 2.6666672.666667 00 00
13.1666713.16667 15.515.5 117.7215117.7215 35.4166735.41667 1.3333331.333333 3.7647053.764705
41.2541.25 39.539.5 95.7575895.75758
17.8333317.83333 1515 84.1121784.11217
평균average 96.807±5.696.807±5.6 25.141±18.925.141±18.9
von frey 자극
(4 g)von frey stimulation
(4 g) 		Vehicle (Hz)Vehicle (Hz) Normalized firing rate (%)Normalized firing rate (%) iN1011-N17 (Hz)iN1011-N17 (Hz) Normalized firing rate (%)Normalized firing rate (%)
BeforeBefore AfterAfter BeforeBefore AfterAfter
30 min30min 5.6111115.611111 5.3333335.333333 95.049595.0495 13.2513.25 11.6666711.66667 88.0503488.05034
12.5833312.58333 12.6666712.66667 100.6623100.6623 10.3888910.38889 8.3333338.333333 80.2138980.21389
6.756.75 8.8333338.833333 130.8642130.8642 4.3333334.333333 1One 23.0769223.07692
26.5833326.58333 25.6666725.66667 96.5517596.55175 4343 31.1666731.16667 72.4806372.48063
44.1666744.16667 51.3333351.33333 116.2264116.2264
20.8333320.83333 20.520.5 98.4000298.40002
평균average 106.292±5.8106.292±5.8 65.955±14.665.955±14.6
60 min60min 5.6111115.611111 5.3333335.333333 95.049595.0495 13.2513.25 9.8333339.833333 74.2138374.21383
12.5833312.58333 11.3333311.33333 90.0662290.06622 10.3888910.38889 66 57.75457.754
6.756.75 6.6666676.666667 98.7654498.76544 4.3333334.333333 0.8333330.833333 19.2307619.23076
26.5833326.58333 23.6666723.66667 89.0282489.02824 4343 3838 88.3720988.37209
44.1666744.16667 5050 113.2075113.2075
20.8333320.83333 16.516.5 79.2000179.20001
평균average 94.219±4.794.219±4.7 59.893±14.959.893±14.9
90 min90min 5.6111115.611111 4.1666674.166667 74.2574374.25743 13.2513.25 7.1666677.166667 54.0880554.08805
12.5833312.58333 1414 111.2583111.2583 10.3888910.38889 2.1666672.166667 20.8556220.85562
6.756.75 77 103.7037103.7037 4.3333334.333333 2.3333332.333333 53.8461553.84615
26.5833326.58333 26.1666726.16667 98.4326398.43263 4343 17.517.5 40.6976740.69767
44.1666744.16667 46.2546.25 104.717104.717
20.8333320.83333 2020 96.0000296.00002
평균average 98.062±5.298.062±5.2 42.372±7.842.372±7.8
120 min120min 5.6111115.611111 6.1666676.166667 109.901109.901 13.2513.25 6.3333336.333333 47.7987447.79874
12.5833312.58333 10.6666710.66667 84.7682684.76826 10.3888910.38889 3.3333333.333333 32.0855532.08555
6.756.75 7.3333337.333333 108.642108.642 4.3333334.333333 0.3333330.333333 7.6923017.692301
26.5833326.58333 24.524.5 92.1630292.16302 4343 1.3333331.333333 3.1007743.100774
44.1666744.16667 48.3333348.33333 109.4339109.4339
20.8333320.83333 20.520.5 98.4000298.40002
평균average 100.551±4.3100.551±4.3 22.669±10.522.669±10.5
von frey 자극
(10 g)von frey stimulation
(10 g) 		Vehicle (Hz)Vehicle (Hz) Normalized firing rate (%)Normalized firing rate (%) iN1011-N17 (Hz)iN1011-N17 (Hz) Normalized firing rate (%)Normalized firing rate (%)
BeforeBefore AfterAfter BeforeBefore AfterAfter
30 min30min 6.1666676.166667 7.57.5 121.6216121.6216 13.5555613.55556 10.833310.8333 79.9180379.91803
14.12514.125 12.1666712.16667 86.1357286.13572 4.3333334.333333 1One 23.0769223.07692
11.4166711.41667 1111 96.3503496.35034 53.9166753.91667 43.1666743.16667 80.0618380.06183
39.7539.75 33.3333333.33333 83.8574383.85743 13.9166713.91667 99 64.6706464.67064
54.6666754.66667 53.1666753.16667 97.256197.2561
22.522.5 22.6666722.66667 100.7408100.7408
평균average 97.660±5.597.660±5.5 61.932±13.561.932±13.5
60 min60min 6.1666676.166667 77 113.5135113.5135 13.5555613.55556 6.8333336.833333 50.4098250.40982
14.12514.125 19.3333319.33333 136.8731136.8731 4.3333334.333333 1.8333331.833333 42.3076942.30769
11.4166711.41667 77 61.3138561.31385 53.9166753.91667 31.1666731.16667 57.8052657.80526
39.7539.75 40.1666740.16667 101.0482101.0482 13.9166713.91667 8.8333338.833333 63.4730463.47304
54.6666754.66667 49.6666749.66667 90.8536690.85366
22.522.5 12.512.5 55.5555655.55556
평균average 93.193±12.793.193±12.7 53.499±4.653.499±4.6
90 min90min 6.1666676.166667 6.6666676.666667 108.1081108.1081 13.5555613.55556 4.6666674.666667 34.4262234.42622
14.12514.125 18.3333318.33333 129.7935129.7935 4.3333334.333333 2.8333332.833333 65.3846165.38461
11.4166711.41667 10.8333310.83333 94.8904594.89045 53.9166753.91667 1919 35.2395735.23957
39.7539.75 35.8333335.83333 90.1467490.14674 13.9166713.91667 12.512.5 89.8203489.82034
54.6666754.66667 53.553.5 97.8658597.86585
22.522.5 20.3333320.33333 90.3703690.37036
평균average 101.863±6.2101.863±6.2 56.218±13.356.218±13.3
120 min120min 6.1666676.166667 6.1666676.166667 100100 13.5555613.55556 88 59.0163759.01637
14.12514.125 16.516.5 116.8142116.8142 4.3333334.333333 0.50.5 11.5384611.53846
11.4166711.41667 11.3333311.33333 99.2700199.27001 53.9166753.91667 1.3333331.333333 2.4729512.472951
39.7539.75 37.8333337.83333 95.1781995.17819 13.9166713.91667 4.54.5 32.3353232.33532
54.6666754.66667 5757 104.2683104.2683
22.522.5 24.3333324.33333 108.1481108.1481
평균average 103.946±3.2103.946±3.2 26.341±12.626.341±12.6
von frey 자극
(100 g)von frey stimulation
(100 g) 		Vehicle (Hz)Vehicle (Hz) Normalized firing rate (%)Normalized firing rate (%) iN1011-N17 (Hz)iN1011-N17 (Hz) Normalized firing rate (%)Normalized firing rate (%)
BeforeBefore AfterAfter BeforeBefore AfterAfter
30 min30min 7.7222227.722222 7.57.5 97.122397.1223 21.1666721.16667 15.6666715.66667 74.0157574.01575
18.1458318.14583 20.6666720.66667 113.8921113.8921 26.526.5 10.8333310.83333 40.8804940.88049
7.257.25 9.1666679.166667 126.4368126.4368 61.0833361.08333 49.8333349.83333 81.5825481.58254
42.5833342.58333 41.8333341.83333 98.2387598.23875 13.0833313.08333 13.013.0 99.3630699.36306
54.8333354.83333 5353 96.6565496.65654
27.1666727.16667 2525 92.0245392.02453
평균average 104.062±5.4104.062±5.4 73.960±12.273.960±12.2
60 min60min 7.7222227.722222 88 103.5971103.5971 21.1666721.16667 14.6666714.66667 69.2913469.29134
18.1458318.14583 17.8333317.83333 98.2778498.27784 26.526.5 16.1666716.16667 61.006361.0063
7.257.25 99 124.1379124.1379 61.0833361.08333 39.8333339.83333 65.2114665.21146
42.5833342.58333 41.1666741.16667 96.6732196.67321 13.0833313.08333 8.58.5 64.9681764.96817
54.8333354.83333 55.555.5 101.2158101.2158
27.1666727.16667 24.524.5 90.1840490.18404
평균average 102.348±4.7102.348±4.7 65.119±1.765.119±1.7
90 min90min 7.7222227.722222 7.1666677.166667 92.8057692.80576 21.1666721.16667 11.3333311.33333 53.5432853.54328
18.1458318.14583 21.1666721.16667 116.6476116.6476 26.526.5 23.1666723.16667 87.421487.4214
7.257.25 8.6666678.666667 119.5402119.5402 61.0833361.08333 15.1666715.16667 24.8294724.82947
42.5833342.58333 37.8333337.83333 88.845488.8454 13.0833313.08333 1010 76.4331476.43314
54.8333354.83333 6363 114.8936114.8936
27.1666727.16667 1919 69.9386469.93864
평균average 100.445±8.1100.445±8.1 60.557±13.860.557±13.8
120 min120min 7.7222227.722222 88 103.5971103.5971 21.1666721.16667 1111 51.968551.9685
18.1458318.14583 21.8333321.83333 120.3215120.3215 26.526.5 16.1666716.16667 61.006361.0063
7.257.25 11.511.5 158.6207158.6207 61.0833361.08333 1.1666671.166667 1.909961.90996
42.5833342.58333 39.1666739.16667 91.9765391.97653 13.0833313.08333 8.1666678.166667 62.420462.4204
54.8333354.83333 63.7563.75 116.2614116.2614
27.1666727.16667 29.3333329.33333 107.9754107.9754
평균average 116.459±9.4116.459±9.4 44.326±14.344.326±14.3
[일반식 2][General Formula 2]
Figure PCTKR2023012835-appb-img-000015
Figure PCTKR2023012835-appb-img-000015
상기 일반식 2에서, C와 D는 하기와 같다.In General Formula 2, C and D are as follows.
C는 비히클(vehicle) 또는 약물 투여 전 활동전위를 발생시키는 최소의 von frey 그램수를나타낸 것이고, D는 비히클(vehicle) 또는 약물 투여 후 활동전위를 발생시키는 최소의 von frey 그램수를 나타낸 것이다.C represents the minimum number of von Frey grams that generate an action potential before administration of the vehicle or drug, and D represents the minimum number of von Frey grams that generate an action potential after administration of the vehicle or drug.
상기 일반식 2에서, Y는 도 5의 결과값을 도출하기 위한 표준화된 활동전위(AP)의 역치값의 변화율(normalized action potential threshold, %)이고, C는 비히클(vehicle) 또는 약물투여 전 활동전위(AP)를 발생시키는 최소의 von frey 그램 수이며, D는 비히클(vehicle) 또는 약물 투여 후 30분, 60분, 90분 및 120분 구간에서 활동전위(AP)를 발생시키는 최소의 von frey 그램 수이다. 위와 같이 도출된 C값과 각각의 D값을 이용하여, 비히클(vehicle) 또는 약물 투여 후 표준화된 활동전위(AP)의 역치값의 변화율(normalized action potential threshold, %)인 Y값을 도출하였다.In the above general formula 2, Y is the rate of change (normalized action potential threshold, %) of the normalized action potential (AP) threshold for deriving the result of Figure 5, and C is the activity before vehicle or drug administration. D is the minimum number of von Frey grams that generate an action potential (AP), and D is the minimum number of von Frey grams that generate an action potential (AP) at 30, 60, 90, and 120 minutes after vehicle or drug administration. It is the number of grams. Using the C value and each D value derived as above, the Y value, which is the normalized action potential threshold (%) change rate of the normalized action potential (AP) threshold after administration of the vehicle or drug, was derived.
구체적으로, 도 5 및 표 6을 참조하면, 전압 의존성 소디움 채널 억제제인 iN1011-N17의 투여군(화학식 1 화합물, 실험군)은 비히클(vehicle)을 투여한 군(대조군)과는 다르게 약물 투여 후 더 큰 자극을 통해서만 신경다발절(DRG)에서 활동전위가 발생됨을 확인하였다. 이러한 결과는 전압 의존성 소디움 채널 억제제인 iN1011-N17 화합물이 활동전위(AP)를 발생시킬 수 있는 자극의 역치값을 높여줌으로써 통증의 억제 효과를 나타낼 수 있다는 점을 증명한다. 따라서 본 발명은 이온채널의 활성을 변화키켜 통증을 억제하는 기전을 갖는 약물 개발과정에서 개발 약물작용의 개념 증명(proof of concept; POC) 및 매카니즘 증명(proof of mechanism; POM) 확보에 어려움을 극복할 수 있는 확실한 대안법으로 판단된다.Specifically, referring to FIG. 5 and Table 6, the group administered iN1011-N17, a voltage-dependent sodium channel inhibitor (compound of formula 1, experimental group), had a larger effect after drug administration, unlike the group administered the vehicle (control group). It was confirmed that action potentials were generated in the nerve bundle ganglion (DRG) only through stimulation. These results demonstrate that the iN1011-N17 compound, a voltage-dependent sodium channel inhibitor, can exert a pain suppressing effect by increasing the threshold value of stimulation that can generate action potentials (AP). Therefore, the present invention overcomes the difficulty in securing proof of concept (POC) and proof of mechanism (POM) of the developed drug action in the process of developing a drug with a mechanism to suppress pain by changing the activity of ion channels. It is judged to be a reliable alternative method.
Vehicle (g)Vehicle (g) Normalized action potential threshold (%)Normalized action potential threshold (%) iN1011-N17 (g)iN1011-N17 (g) Normalized action potential threshold (%)Normalized action potential threshold (%)
BeforeBefore AfterAfter BeforeBefore AfterAfter
30 min30min 0.160.16 0.160.16 100100 0.160.16 0.40.4 250250
0.60.6 0.60.6 100100 0.070.07 0.40.4 571.4286571.4286
0.60.6 0.60.6 100100 44 1010 250250
0.040.04 0.020.02 5050 0.60.6 1One 166.6667166.6667
0.160.16 0.160.16 100100 0.040.04 0.070.07 175175
평균average 90±10.090±10.0 282.619±74.4282.619±74.4
60 min60min 0.160.16 0.160.16 100100 0.160.16 0.40.4 250250
0.60.6 0.60.6 100100 0.070.07 0.40.4 571.4286571.4286
0.60.6 0.60.6 100100 44 1010 250250
0.040.04 0.020.02 5050 0.60.6 1One 166.6667166.6667
0.160.16 0.160.16 100100 0.040.04 0.070.07 175175
평균average 90±10.090±10.0 282.619±74.4282.619±74.4
90 min90min 0.160.16 0.160.16 100100 0.160.16 0.40.4 250250
0.60.6 0.40.4 66.6666766.66667 0.070.07 0.160.16 228.5714228.5714
0.60.6 0.60.6 100100 44 1010 250250
0.040.04 0.020.02 5050 0.60.6 1One 166.6667166.6667
0.160.16 0.160.16 100100 0.040.04 0.070.07 175175
평균average 83.333±10.583.333±10.5 214.048±18.1214.048±18.1
120 min120min 0.160.16 0.160.16 100100 0.160.16 0.40.4 250250
0.60.6 0.40.4 66.6666766.66667 0.070.07 0.40.4 571.4286571.4286
0.60.6 0.60.6 100100 44 1010 250250
0.040.04 0.020.02 5050 0.60.6 1One 166.6667166.6667
0.160.16 0.160.16 100100 0.040.04 0.40.4 10001000
평균average 83.333±10.583.333±10.5 447.619±154.5447.619±154.5
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 관련 기술 분야의 통상의 지식을 가진 자에게 있어 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구범위와 그의 등가물에 의하여 정의될 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

  1. 인비보(in-vivo) 세포외(extracellular) 신경다발절(DRG) 기록법을 이용하여 전압 의존성 소디움 채널 억제제가 소디움 채널의 활성을 선택적으로 억제함으로써 세포막 전위가 활동전위를 유발하기 위한 역치(threshold)에 도달하는 것을 억제하거나 혹은 지연시켜 활동전위(AP)의 발생 빈도를 줄여주는 기전의 약물에 대한 약효를 평가하는 전기생리학적 약효 평가법에 있어서,Using in-vivo extracellular nerve ganglion (DRG) recording, a voltage-dependent sodium channel inhibitor selectively inhibits the activity of sodium channels, thereby determining the threshold for the cell membrane potential to trigger an action potential. In the electrophysiological drug efficacy evaluation method that evaluates the efficacy of drugs with a mechanism that reduces the frequency of action potentials (AP) by inhibiting or delaying their arrival,
    (S1) 비히클(vehicle)을 투여하여 대조군 발화율(control firing rate) 구간의 활동전위 표준화된 발화율(normalized firing rate, %)를 측정하는 단계;(S1) measuring the normalized firing rate (%) of action potentials in the control firing rate section by administering a vehicle;
    (S2) 전압 의존성 소디움 채널 억제제를 투여하여 약물 발화율(firing rate) 구간의 활동전위 표준화된 발화율(normalized firing rate, %)를 측정하는 단계; 및(S2) measuring the normalized firing rate (%) of action potentials in the drug firing rate section by administering a voltage-dependent sodium channel inhibitor; and
    (S3) (S1)에서 측정된 활동전위 표준화된 발화율 대비 (S2)에서 측정된 활동전위 표준화된 발화율의 억제 정도를 통계적으로 확인하는 단계를 포함하고,(S3) comprising the step of statistically confirming the degree of suppression of the action potential normalized firing rate measured in (S2) compared to the action potential normalized firing rate measured in (S1),
    상기 (S1) 및 (S2)에서 활동전위 표준화된 발화율(normalized firing rate, %)은 하기 일반식 1을 통해 측정하는 것을 특징으로 하는 약효 평가법:A drug efficacy evaluation method characterized in that the action potential normalized firing rate (%) in (S1) and (S2) is measured using the following general formula 1:
    [일반식 1][General Formula 1]
    Figure PCTKR2023012835-appb-img-000016
    Figure PCTKR2023012835-appb-img-000016
    상기 일반식 1에서, X는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 후 활동전위 표준화된 발화율(normalized firing rate, %)이고, A는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 전 각각의 von frey 그램 자극에 반응하여 발생되는 활동전위 발생 빈도(Hz)이며, B는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 직후인 전압 의존성 소디움 채널 억제제 발화율(firing rate) 구간의 각각의 von frey 그램 자극에 반응하여 발생되는 활동전위 발생 빈도(Hz)이다.In the above general formula 1, B is the action potential generation frequency (Hz) generated in response to von Frey gram stimulation, and B is each von Frey gram in the voltage-dependent sodium channel inhibitor firing rate section immediately after administration of vehicle or voltage-dependent sodium channel inhibitor. This is the frequency (Hz) of action potentials generated in response to stimulation.
    Figure PCTKR2023012835-appb-img-000017
    ,
    Figure PCTKR2023012835-appb-img-000018
    .
    Figure PCTKR2023012835-appb-img-000017
    ,
    Figure PCTKR2023012835-appb-img-000018
    .
  2. 제1항에 있어서, 상기 약효 평가법은 하기 조건을 만족하는 것을 특징으로 하는 약효 평가법:The drug efficacy evaluation method according to claim 1, wherein the drug efficacy evaluation method satisfies the following conditions:
    조건 1: 상기 일반식 1에서, 전압 의존성 소디움 체널 억제제를 투여한 경우, B 값은 A 값보다 작음 (B 값 < A 값)Condition 1: In the general formula 1 above, when a voltage-dependent sodium channel inhibitor is administered, the B value is less than the A value (B value < A value)
    조건 2: 상기 일반식 1에서, (S2)에서 측정된 활동전위 표준화된 발화율은 (S1)에서 측정된 활동전위 표준화된 발화율보다 작으며, 그 차이가 통계적으로 유의미한 경우임. Condition 2: In General Formula 1 above, the action potential normalized firing rate measured in (S2) is smaller than the action potential normalized firing rate measured in (S1), and the difference is statistically significant.
  3. 제1항에 있어서, 상기 전압 의존성 소디움 채널 억제제는 하기 화학식 1로 표시되는 화합물인 것인 약효 평가법:The drug efficacy evaluation method according to claim 1, wherein the voltage-dependent sodium channel inhibitor is a compound represented by the following formula (1):
    [화학식 1][Formula 1]
    Figure PCTKR2023012835-appb-img-000019
    Figure PCTKR2023012835-appb-img-000019
  4. 제1항에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 1 mg/kg 내지 100 mg/kg를 투여하는 것인 약효 평가법.The drug efficacy evaluation method according to claim 1, wherein 1 mg/kg to 100 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, is administered.
  5. 제4항에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 30 mg/kg을 투여하는 것인 약효 평가법.The drug efficacy evaluation method according to claim 4, wherein 30 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, is administered.
  6. 제5항에 있어서, 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 30 mg/kg을 투여할 때 상기 일반식 1에서, X, A 및 B는 하기와 같은 것인 약효 평가법:The drug efficacy evaluation method according to claim 5, wherein when 30 mg/kg of the compound represented by Formula 1, which is a voltage-dependent sodium channel inhibitor, is administered, in Formula 1, X, A and B are as follows:
    X: 0 % 내지 99.36306 %X: 0% to 99.36306%
    A: 1.583333 내지 61.08333 HzA: 1.583333 to 61.08333 Hz
    B: 0 내지 49.83333 Hz.B: 0 to 49.83333 Hz.
  7. 제1항에 있어서, 상기 약효 평가법은, (S1) 단계 이전에 설치류 마취 후 척수(spinal cord)의 특정 타겟 부위에서 세포외 기록법을 수행하기 위해 척수 클램퍼(spinal cord clamper)를 이용하여 척추뼈를 고정하는 단계를 포함하는 약효 평가법.According to claim 1, the drug efficacy evaluation method uses a spinal cord clamper to perform extracellular recording in a specific target area of the spinal cord after anesthetizing a rodent before step (S1). Drug efficacy evaluation method including a fixation step.
  8. 제1항에 있어서, 상기 von frey 그램은 1 내지 100 g인 것을 특징으로 하는 약효 평가법.The drug efficacy evaluation method according to claim 1, wherein the von Frey gram is 1 to 100 g.
  9. 인비보(in-vivo) 세포외(extracellular) 신경다발절(DRG) 기록법을 이용하여 전압 의존성 소디움 채널 억제제가 소디움 채널의 활성을 선택적으로 억제함으로써 세포막 전위가 활동전위를 유발하기 위한 역치(threshold)에 도달하는 것을 억제하거나 혹은 지연시켜 활동전위의 발생 빈도를 줄여주는 기전의 약물에 대한 약효를 평가하는 전기생리학적 약효 평가법에 있어서,Using in-vivo extracellular nerve ganglion (DRG) recording, a voltage-dependent sodium channel inhibitor selectively inhibits the activity of sodium channels, thereby determining the threshold for the cell membrane potential to trigger an action potential. In an electrophysiological drug efficacy evaluation method that evaluates the efficacy of drugs with a mechanism that reduces the frequency of action potentials by inhibiting or delaying their arrival,
    (S1') 비히클(vehicle)을 투여하여 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)을 측정하는 단계;(S1') measuring the normalized action potential threshold change rate (%) by administering a vehicle;
    (S2') 전압 의존성 소디움 채널 억제제를 투여하여 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)을 측정하는 단계; 및(S2') measuring the normalized action potential threshold change rate (%) by administering a voltage-dependent sodium channel inhibitor; and
    (S3') (S1')에서 측정된 표준화된 활동전위의 역치값의 변화율 대비 (S2')에서 측정된 표준화된 활동전위의 역치값의 변화율의 증가 정도를 통계적으로 확인하는 단계를 포함하고,(S3') Statistically confirming the degree of increase in the rate of change in the threshold value of the standardized action potential measured in (S2') compared to the rate of change in the threshold value of the standardized action potential measured in (S1'),
    상기 (S1') 및 (S2')에서 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)은 하기 일반식 2를 통해 측정하는 것을 특징으로 하는 약효 평가법:In (S1') and (S2'), the rate of change (normalized action potential threshold, %) of the normalized action potential threshold is a drug efficacy evaluation method characterized in that it is measured using the following general formula 2:
    [일반식 2][General Formula 2]
    Figure PCTKR2023012835-appb-img-000020
    Figure PCTKR2023012835-appb-img-000020
    상기 일반식 2에서, Y는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 후 표준화된 활동전위의 역치값의 변화율(normalized action potential threshold, %)이고, C는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 전 활동전위를 발생시키는 최소의 von frey 그램 수이며, D는 비히클(vehicle) 또는 전압 의존성 소디움 채널 억제제 투여 후 활동전위를 발생시키는 최소의 von frey 그램 수이다.In General Formula 2, Y is the normalized action potential threshold change rate (%) after administration of vehicle or voltage-dependent sodium channel inhibitor, and C is vehicle or voltage-dependent sodium channel D is the minimum number of von Frey grams that generate an action potential before administration of the inhibitor, and D is the minimum number of von Frey grams that generate an action potential after administration of a vehicle or voltage-dependent sodium channel inhibitor.
  10. 제9항에 있어서, 상기 약효 평가법은 하기 조건을 만족하는 것을 특징으로 하는 약효 평가법:The drug efficacy evaluation method according to claim 9, wherein the drug efficacy evaluation method satisfies the following conditions:
    조건 3: 상기 일반식 2에서, 전압 의존성 소디움 체널 억제제를 투여한 경우, D 값은 C 값보다 큼Condition 3: In General Formula 2 above, when a voltage-dependent sodium channel inhibitor is administered, the D value is greater than the C value.
    조건 4: 상기 일반식 2에서, (S2')에서 측정된 표준화된 활동전위의 역치값의 변화율은 (S1')에서 측정된 표준화된 활동전위의 역치값의 변화율보다 크며, 그 차이가 통계적으로 유의미한 경우임.Condition 4: In the above general formula 2, the rate of change of the threshold value of the standardized action potential measured in (S2') is greater than the rate of change of the threshold value of the standardized action potential measured in (S1'), and the difference is statistically This is a significant case.
  11. 제9항에 있어서, 상기 전압 의존성 소디움 채널 억제제는 하기 화학식 1로 표시되는 화합물인 것인 약효 평가법:The drug efficacy evaluation method according to claim 9, wherein the voltage-dependent sodium channel inhibitor is a compound represented by the following formula (1):
    [화학식 1][Formula 1]
    Figure PCTKR2023012835-appb-img-000021
    Figure PCTKR2023012835-appb-img-000021
  12. 제9항에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 1 mg/kg 내지 100 mg/kg를 투여하는 것인 약효 평가법.The drug efficacy evaluation method according to claim 9, wherein 1 mg/kg to 100 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, is administered.
  13. 제12항에 있어서, 상기 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 30 mg/kg을 투여하는 것인 약효 평가법.The drug efficacy evaluation method according to claim 12, wherein 30 mg/kg of the compound represented by Formula 1, which is the voltage-dependent sodium channel inhibitor, is administered.
  14. 제13항에 있어서, 전압 의존성 소디움 채널 억제제인 화학식 1로 표시되는 화합물을 30 mg/kg을 투여할 때 상기 일반식 2에서, Y, C 및 D는 하기와 같은 것인 약효 평가법:The drug efficacy evaluation method according to claim 13, wherein when 30 mg/kg of the compound represented by Formula 1, which is a voltage-dependent sodium channel inhibitor, is administered, in Formula 2, Y, C and D are as follows:
    Y: 50 % 내지 1000 %Y: 50% to 1000%
    C: 0.04 내지 4 gC: 0.04 to 4 g
    D: 0.02 내지 10 g.D: 0.02 to 10 g.
  15. 제9항에 있어서, 상기 약효 평가법은, (S1') 단계 이전에 설치류 마취 후 척수(spinal cord)의 특정 타겟 부위에서 세포외 기록법을 수행하기 위해 척수 클램퍼(spinal cord clamper)를 이용하여 척추뼈를 고정하는 단계를 포함하는 약효 평가법.The method of claim 9, wherein the drug efficacy evaluation method uses a spinal cord clamper to perform extracellular recording in a specific target area of the spinal cord after anesthetizing a rodent before the (S1') step. A drug efficacy evaluation method including the step of fixing.
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