Drug screening method

Abstract

To provide a drug screening method that allows for confirming an effect of a test target substance on an expression level or a function of at least one of Nav1.7, Nav1.8, and Nav1.9, while reducing burden on animals relative to conventional screening methods.SOLUTION: A drug screening method according to the present invention increases or decreases an expression level of at least one selected from Nav1.7, Nav1.8, and Nav1.9, or enhances or reduces a function of the at least one. The method comprises the steps of: administering a test target substance to a non-human animal that expresses at least one selected from Nav1.7, Nav1.8, and Nav1.9 and a photosensitive cation channel in dorsal root ganglion neurons; irradiating, after administration of the test target substance, the non-human animal with light having a wavelength at which the photosensitive cation channel opens; and confirming whether or not the non-human animal escapes from the irradiated light.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening drugs. [Background technology]

[0002] Diseases associated with voltage-gated sodium channels have been known, one of which is neuropathic pain.

[0003] Neuropathic pain occurs due to damage to sensory nerves and is closely related to excitation of voltage-gated sodium channel 1.7 (hereinafter also referred to as "Nav1.7"), voltage-gated sodium channel 1.8 (hereinafter also referred to as "Nav1.8"), or voltage-gated sodium channel 1.9 (hereinafter also referred to as "Nav1.9"), which are mainly expressed in dorsal root ganglion neurons.

[0004] To screen for drugs effective against neuropathic pain, a possible approach is to administer a test substance (a substance that can be a drug candidate) to a non-human animal model of neuropathic pain and observe its effects.

[0005] For example, Patent Document 1 describes that a rat model of neuropathic pain was produced by making a midline incision in the back of a rat, cutting the L6 transverse process, and then ligating the L5 lumbar nerve with silk thread. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2020-026397 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the method described in Patent Document 1 has the problem that it places a heavy burden on the body of the animal used as a neuropathic pain model.

[0008] In addition, when using conventional neuropathic pain models, after administering the test substance, the model animals must be euthanized, and the dorsal root ganglia must be removed to confirm the expression levels of Nav1.7, Nav1.8, or Nav1.9. From the perspective of the 3Rs (Replacement, Reduction, Refinement) related to animal testing, there is a need to develop screening methods that place less strain on model animals.

[0009] In light of these circumstances, the present invention aims to provide a drug screening method that can confirm the effect of a test substance on the expression level or function of at least one of Nav1.7, Nav1.8, and Nav1.9 while reducing the burden on animals compared to conventional screening methods. [Means for solving the problem]

[0010] One aspect of the present invention includes the following aspects. [1] A method for screening for a drug that increases or decreases the expression level of at least one of voltage-gated sodium channels 1.7 (Nav1.7), 1.8 (Nav1.8), and 1.9 (Nav1.9), or enhances or decreases the function of said at least one, comprising the steps of administering a test substance to a non-human animal that expresses at least one of Nav1.7, 1.8, and 1.9 and a light-sensitive cation channel in dorsal root ganglion neurons; A drug screening method comprising the steps of: irradiating the non-human animal after administration of a test substance with light of a wavelength at which the light-sensitive cation channel opens; and confirming whether the non-human animal escapes from the irradiated light.

[0011] [2] The drug screening method according to [1], further comprising the step of increasing the pain sensitivity of the non-human animal prior to the step of administering the test substance.

[0012] [3] The drug screening method according to [2], wherein the step of increasing the sensitivity involves irradiating the cells with light for 30 minutes or more.

[0013] [4] The drug screening method of claim 2, wherein the step of increasing sensitivity involves administering to the non-human animal a compound selected from the group consisting of FK506, streptozotocin, paclitaxel, cisplatin, oxaliplatin, and vincristine.

[0014] [5] The drug screening method according to any one of [1] to [4], wherein the light-sensitive cation channel is channelrhodopsin 2.

[0015] [6] A drug screening method according to any one of [1] to [5], wherein in the step of confirming whether or not the non-human animal escapes from the light, the ratio of the number of times the non-human animal escapes from the light to the number of times the light is irradiated is confirmed.

[0016] [7] A drug screening method described in [6], in which if the percentage of times the subject escapes from the light is lower than that of a control group not administered the test substance, the test substance is selected as a candidate drug that reduces the expression level or function of at least one of Nav1.7, 1.8 and 1.9.

[0017] [8] A drug screening method described in [6], in which if the percentage of times the subject escapes from the light is higher than that of a control group not administered the test substance, the test substance is selected as a candidate drug that increases the expression level or enhances the function of at least one of Nav1.7, 1.8 and 1.9.

[0018] [9] A drug screening method described in any one of [1] to [5], wherein in the step of confirming whether or not the non-human animal escapes from the light, the time the non-human animal spends in the first space is measured in an environment in which the non-human animal can move freely between a first space to which the light is irradiated and a second space to which the light is not irradiated.

[0019]

[10] A drug screening method described in [9], in which the test substance is selected as a candidate drug that reduces the expression level or function of at least one of Nav1.7, 1.8 and 1.9 if the test substance spends a longer time in the first space than a control group that has not been administered the test substance.

[0020]

[11] A drug screening method described in [9], in which the test substance is selected as a candidate drug that increases the expression level or enhances the function of at least one of Nav1.7, 1.8 and 1.9 if the time spent in the first space is shorter than that of a control group not administered the test substance. [Effects of the Invention]

[0021] According to the present invention, it is possible to screen for drugs that can confirm the effect of a test substance on the expression level or function of at least one of Nav1.7, Nav1.8, and Nav1.9 while reducing the burden on animals compared to conventional screening methods. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram showing the generation of non-human animals that co-express at least one of Nav1.7, 1.8, and 1.9 channels and a light-sensitive cation channel in dorsal root ganglion neurons by breeding. [Figure 2] FIG. 1 is a schematic diagram showing how a non-human animal that has undergone a test substance administration step is irradiated multiple times with light of a wavelength that opens a light-gated cation channel. [Figure 3] FIG. 10 is a schematic perspective view showing a specific example of checking whether a non-human animal escapes from light irradiated in an irradiation step. [Figure 4] 1 is a graph showing the relationship between the ratio of the number of times escape behavior occurred to the total number of times light was irradiated and the intensity of blue light in mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons. [Figure 5]10 is a graph showing the results of an OPA test in which FK506 was administered to mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons. [Figure 6] 10 is a graph showing the results of administering FK506 to mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons, and measuring the expression levels of Nav1.7 mRNA and β-actin mRNA in dorsal root ganglion neurons. [Figure 7] 1 is a graph showing the results of a von Frey test performed on mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons after a pain sensitivity increasing procedure. [Figure 8] 1 is a graph showing the results of a von Frey test performed on mice expressing Nav1.8 and channelrhodopsin 2 in dorsal root ganglion neurons after a pain sensitivity increasing procedure. [Figure 9] 1 is a graph showing the results of a von Frey test performed on mice expressing Nav1.9 and channelrhodopsin 2 in dorsal root ganglion neurons after a pain sensitivity increasing procedure. DETAILED DESCRIPTION OF THE INVENTION

[0023] Preferred embodiments of the present invention will now be described in detail.

[0024] [Drug screening method] A preferred embodiment of the present invention provides a drug screening method for drugs that increase or decrease the expression level of at least one of voltage-gated sodium channels 1.7 (Nav1.7), 1.8 (Nav1.8), and 1.9 (Nav1.9), or enhance or decrease the function of at least one of the voltage-gated sodium channels. The method includes the steps of administering a test substance to a non-human animal that expresses at least one of Nav1.7, 1.8, and 1.9 and a light-sensitive cation channel in dorsal root ganglion neurons (test substance administration step), irradiating the non-human animal with light of a wavelength at which the light-sensitive cation channel opens (irradiation step), and confirming whether the non-human animal escapes from the irradiated light (confirmation step). The function of the voltage-gated sodium channel is to selectively allow sodium ions to permeate into neurons, thereby generating an action potential or facilitating the generation of an action potential.

[0025] <Non-human animals> The non-human animal may be any mammal that expresses at least one of Nav1.7, 1.8, and 1.9 and a light-sensitive cation channel in dorsal root ganglion neurons, and is not particularly limited to, but examples include mice, rats, rabbits, guinea pigs, hamsters, hedgehogs, dogs, cats, monkeys, horses, cows, pigs, sheep, etc.

[0026] (light-sensitive cation channel) The light-sensitive cation channels expressed in dorsal root ganglion neurons of non-human animals are not particularly limited as long as they are light-sensitive cation channels found in Chlamydomonas or other organisms or modified forms thereof, but examples include channelrhodopsin 1 (ChR1) and channelrhodopsin 2 (ChR2).

[0027] At least one of the Nav1.7, 1.8, and 1.9 channels, which are expressed in non-human animals along with light-sensitive cation channels, is a voltage-gated sodium channel whose expression level is increased or decreased, or whose function is enhanced or decreased, by the drug to be screened (screening result).

[0028] The method for expressing at least one of Nav1.7, 1.8, and 1.9 channels and a light-sensitive cation channel in dorsal root ganglion neurons is not particularly limited, but an example of a method for co-expressing at least one of Nav1.7, 1.8, and 1.9 channels and a light-sensitive cation channel in dorsal root ganglion neurons is described in detail below.

[0029] FIG. 1 is a schematic diagram showing the process of breeding to generate non-human animals that co-express at least one of Nav1.7, 1.8, and 1.9 channels and a light-gated cation channel in dorsal root ganglion neurons.

[0030] FIG. 1 shows an example of the production of a non-human animal (a mouse as an example) that co-expresses channelrhodopsin 2, an example of a light-sensitive cation channel, together with at least one of Nav1.7, 1.8, and 1.9 channels.

[0031] As shown in Figure 1, one of the parents to be crossed contains a gene encoding iCre recombinase (a codon-improved CRE recombinase) downstream of a gene encoding a voltage-gated sodium channel (Nav1.x, x = 7-9). The other parent contains a stop codon between two loxP sequences, and a gene encoding channelrhodopsin 2 downstream of that. The CAG promoter is used to control the transcription of channelrhodopsin 2.

[0032] The offspring of non-human animals obtained by mating these parents has, in one allele, a gene encoding iCre recombinase downstream of Nav1.x, and in the other allele, a stop codon between two loxP sequences and a gene encoding channelrhodopsin 2 further downstream.

[0033] In offspring non-human animals, cells that do not express Nav1.x (x = 7-9) do not express iCre recombinase, and channelrhodopsin 2 is not expressed because there is a stop codon between the gene encoding channelrhodopsin 2 and the CAG promoter.

[0034] In contrast, cells expressing Nav1.x (x = 7-9) (mainly dorsal root ganglion neurons) express iCre recombinase along with Nav1.x, which excises the STOP codon between the two loxP sequences. As a result, Nav1.x-expressing cells also express channelrhodopsin 2.

[0035] Furthermore, by mating offspring that are heterozygous for an allele containing a gene encoding iCre recombinase downstream of Nav1.x (x = 7-9) with offspring that are heterozygous for an allele containing channelrhodopsin 2, it is possible to produce non-human animals that are homozygous for both alleles. Note that Cre recombinase can also be used instead of iCre recombinase.

[0036] Non-human animals that co-express Nav1.x (x=7-9) and a light-sensitive cation channel are also described in the following literature, and such non-human animals themselves can be obtained by known techniques. T. Maruta et al., Selective optogenetic activation of NaV1.7-expressing afferents in NaV1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuli. PLoS One, 17 (10) 2022.

[0037] The above describes in detail a method for expressing at least one of Nav1.7, 1.8, and 1.9 and a light-sensitive cation channel in dorsal root ganglion neurons in a non-human animal. However, the genetic design of a non-human animal that expresses at least one of Nav1.7, 1.8, and 1.9 and a light-sensitive cation channel in dorsal root ganglion neurons is not limited to this.

[0038] <Test substance administration step> (Test substance) The test substance (a substance that can be a candidate drug) that has the potential to increase or decrease the expression level of at least one of Nav1.7, 1.8, and 1.9, or to enhance or decrease the function of said at least one, i.e., the test substance to be administered to the non-human animal described above, is not particularly limited, but examples thereof include proteins, peptides, nucleic acids, antibodies, and low molecular weight compounds with a molecular weight of 1,000 or less.

[0039] Furthermore, natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, etc. may be used as test substances.

[0040] The test substance may be administered to the non-human animal in combination with a pharmaceutically acceptable carrier or other ingredients. A "pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the test substance and does not exhibit substantial toxicity to the subject to which it is administered.

[0041] The type of pharmaceutically acceptable carrier is not particularly limited, and examples thereof include solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc. One type of pharmaceutically acceptable carrier may be used alone, or two or more types may be used in combination.

[0042] The term "not substantially toxic" means that the component does not exhibit toxicity to the non-human animal to which it is administered at a dose normally used.

[0043] In the drug screening method of this embodiment, not inhibiting the physiological activity of the test substance more specifically means not inhibiting the activity of increasing or decreasing the expression or function of Nav1.7, 1.8, and 1.9.

[0044] Examples of the other ingredients include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, coloring agents, and the like.

[0045] (Administration route of test substance) The route of administration of a test substance to a non-human animal is not particularly limited, and the test substance can be administered orally or parenterally. Whether to administer the test substance or not can be determined appropriately by a person skilled in the art depending on the type of non-human animal and the type of test substance.

[0046] Examples of parenteral administration include intravenous administration, intranasal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and enteral administration.

[0047] (Test substance dosage) The dose of the test substance is not particularly limited, and may be determined appropriately depending on, for example, the type and weight of the non-human animal, the type of test substance, the method of administration, and the like.

[0048] (Number of times test substance is administered) The test substance may be administered once or repeatedly. In the case of repeated administration, the administration interval is not particularly limited, but examples include every few hours, 2-3 times a day, once a day, once every 2-3 days, once a week, etc.

[0049] <Irradiation process> The wavelength of the light irradiated in the irradiation step is the wavelength at which the light-sensitive cation channel opens, in other words, the wavelength at which the light-sensitive cation channel is activated. The light at which the light-sensitive cation channel opens may be, for example, visible light.

[0050] When the light-sensitive cation channel expressed in dorsal root ganglion neurons of a non-human animal is, for example, channelrhodopsin 1, the light irradiated in the irradiation step is preferably green light with a wavelength of around 500 nm (for example, 490 to 510 nm).

[0051] Furthermore, when the light-sensitive cation channel expressed in dorsal root ganglion neurons of a non-human animal is channelrhodopsin 2, the light irradiated in the irradiation step is preferably blue light with a wavelength of around 470 nm (for example, 460 to 480 nm).

[0052] The intensity of the light irradiated in the irradiation step is not particularly limited, but may be, for example, 0.7 to 6 mW in the plantar irradiation test described in detail below, or 7 to 10 mW in the OPA test described in detail below. The light intensity can be determined appropriately depending on the type, strain, and size of the non-human animal.

[0053] The irradiation step is preferably carried out after the above-mentioned test substance administration step in order to confirm the effect of administering the test substance. When the test substance is administered repeatedly, the irradiation step may be carried out at any time after the first administration, or may be carried out after the completion of the repeated administration. The irradiation step may be carried out after a period of time expected for the effect of the test substance to appear after the test substance administration step is carried out. The period of time expected for the effect of the test substance to appear varies depending on the type of test substance, the expected mechanism of action, etc.

[0054] When a systemic promoter (expressed throughout the body) such as the CAG promoter is used as the promoter controlling the transcription of channelrhodopsin 2, any part of the skin surface of the non-human animal may be used, but a part that is not covered with hair (having little hair) is preferred. Such parts include the soles of the hands and feet (palms and soles).

[0055] When light of a wavelength that opens the light-sensitive cation channel is irradiated onto the skin of the non-human animal, the light is received by receptors at the terminals of dorsal root ganglion neurons that express the light-sensitive cation channel. This causes depolarization in the dorsal root ganglion neurons, similar to when Nav1.x (x = 7-9) is opened, generating an action potential. As a result, pain sensory information is transmitted to the spinal cord, and the non-human animal feels pain at the site exposed to the light.

[0056] <Confirmation process> In the confirmation step, it is confirmed whether the non-human animal escapes (in other words, avoids or abhors) the light (in other words, pain) irradiated in the irradiation step. For this reason, the confirmation step is carried out after the start of the irradiation step.

[0057] An example of the light-avoidance behavior of a non-human animal is the behavior of moving at least the irradiated body part away from the irradiated light.

[0058] Examples of behaviors that move at least an irradiated body part away from the irradiated location include, for example, retracting or moving the irradiated hand or foot when light is irradiated onto the hand or foot, and also include non-human animals moving away from the irradiated location (moving away from the irradiated location).

[0059] Here, if the administration of the test substance increases the expression level or function of at least one of Nav1.7, 1.8, and 1.9, the membrane potential (amount of current) will be higher than before the increase in expression level or function, even when the same amount and intensity of light is received by the above receptors on the skin surface.

[0060] As a result, the non-human animal feels stronger pain than when the test substance is not administered, and as a result, the animal exhibits the above-mentioned behavior of avoiding light (pain) more frequently (in other words, more frequently) during or after light irradiation than when the test substance is not administered.

[0061] In contrast, when the administration of a test substance reduces the expression or function of at least one of Nav1.7, 1.8, and 1.9, the membrane potential (amount of current) is reduced compared to before the increase in expression or function, even when the same amount and intensity of light is received by the receptors on the skin surface. Therefore, the probability of exhibiting the above-mentioned behavior of avoiding light (pain) during or after light irradiation is reduced compared to when the test substance is not administered.

[0062] From the above, by confirming whether or not the subject avoids the light (in other words, pain) irradiated in the irradiation step, it is possible to determine whether or not the test substance administered in the test substance administration step has the activity of increasing or decreasing the expression level of at least one of Nav1.7, 1.8, and 1.9, or enhancing or decreasing its function.

[0063] For example, if administration of a test substance to a non-human animal that expresses at least one of Nav1.7, 1.8, and 1.9 and a light-sensitive cation channel in dorsal root ganglion neurons results in significantly more escape behavior during or after the subsequent exposure to light than in a control group that has not been administered the test substance, the test substance is found to have the activity of increasing the expression level or function of one or more voltage-gated sodium channels expressed in dorsal root ganglion neurons.

[0064] Therefore, the test substance can be selected as a candidate drug that increases the expression level or function of one or more voltage-gated sodium channels expressed in dorsal root ganglion neurons.

[0065] In contrast, if administration of a test substance to a non-human animal that expresses at least one of Nav1.7, 1.8, and 1.9 and a light-sensitive cation channel in dorsal root ganglion neurons results in significantly less escape behavior during or after the subsequent exposure to light than in a control group that has not been administered the test substance, the test substance is found to have the activity of reducing the expression level or function of one or more voltage-gated sodium channels expressed in dorsal root ganglion neurons.

[0066] Therefore, the test substance can be selected as a candidate drug that reduces the expression level or function of one or more voltage-gated sodium channels expressed in dorsal root ganglion neurons. The results from the control group may be results obtained using non-human animals before administration of the test substance. In other words, it is possible to confirm whether the same non-human animals avoid the light irradiated in the irradiation step before and after administration of the test substance, and compare the results before and after administration. Below, a plantar irradiation test, which is an example of confirming whether a non-human animal avoids the light irradiated in the irradiation step, is described in detail.

[0067] FIG. 2 is a schematic diagram showing how a non-human animal that has undergone a test substance administration step is irradiated multiple times with light of a wavelength that opens a light-gated cation channel.

[0068] In this example, a non-human animal that has undergone the test substance administration step is placed on a glass plate 5, and using a light irradiation device 6 such as an LED, the above-mentioned light that opens the light-sensitive cation channels is irradiated from below through the glass plate 5 multiple times onto the soles of the non-human animal's hind paws (irradiation step). Then, the proportion of times the non-human animal escapes from the light (pulls back its hind legs) out of the total number of times irradiation is confirmed (calculated) (confirmation step). In addition, a control group using non-human animals that have not undergone the test substance administration step (not administered the test substance) is also prepared.

[0069] The interval between light irradiations is preferably set to a time (for example, 30 seconds or more) that is longer than the time it takes for a non-human animal to retract its hind legs in response to the irradiated light and then return them to a position close to their original position.

[0070] When the proportion of times the non-human animal escapes from the light out of the total number of times irradiation was confirmed and the proportion of times the non-human animal escapes from the light after the test substance administration step is found to be significantly lower than the proportion of times the non-human animals in the control group escaped from the light (in other words, the number of times they escaped from the light is significantly less), the test substance administered in the test substance administration step can be selected as a candidate drug that reduces the expression level or function of at least one of Nav1.7, 1.8, and 1.9 expressed in dorsal root ganglion neurons in the non-human animal.

[0071] In contrast, when the proportion of times the non-human animal escapes from light out of the total number of times irradiation was confirmed, if the proportion of times the non-human animal escapes from light after the test substance administration step is significantly higher than the proportion of times the non-human animals in the control group escaped from light (in other words, the number of times they escaped from light is significantly higher), the test substance administered in the test substance administration step can be selected as a candidate drug that increases the expression level or enhances the function of at least one of Nav1.7, 1.8, and 1.9 expressed in dorsal root ganglion neurons in non-human animals.

[0072] In the example shown in Figure 2, light was irradiated onto the soles of the non-human animal's feet, but the area to which light is irradiated is not limited to the soles of the feet, and may be any area with little hair.

[0073] Above, we have described in detail one specific example (plantar irradiation test) of confirming whether or not a non-human animal will escape from the light irradiated in the irradiation process. Below, we will explain the OPA test, which is another example of confirming whether or not a non-human animal will escape from the light irradiated in the irradiation process.

[0074] FIG. 3 is a schematic perspective view showing a specific example of checking whether or not a non-human animal escapes from the light irradiated in the irradiation step.

[0075] As shown in Figure 3, first, the non-human animal 3 that has undergone the test substance administration step is placed in a container 10 that includes a first space 1 that is irradiated with the light that opens the light-sensitive cation channels, and a second space 2 that is not irradiated with the light. The container 10 is designed so that the non-human animal 3 can move freely between the first space 1 and the second space 2. The floor 10a of the container 10 is transparent so that the light that opens the light-sensitive cation channels can pass through. In addition, a control group is also prepared by placing non-human animals that have not undergone the test substance administration step (i.e., have not been administered the test substance) in the container 10.

[0076] After the non-human animal 3 is placed in the container 10, while the non-human animal 3 is in the first space 1, the soles (plantar surfaces) of the hind feet of the non-human animal 3 are irradiated from below with the light that opens the light-sensitive cation channels using a light irradiation device 6 such as an LED arranged below the floor surface 10a (irradiation step). When the light-sensitive cation channel expressed in the dorsal root ganglion neurons of the non-human animal 3 is, for example, channelrhodopsin 2, the irradiated light is blue light with a wavelength of around 470 nm. Furthermore, while the non-human animal is in the second space 2, light other than blue (for example, green) is irradiated from below onto the soles of the hind feet of the non-human animal 3, or no light is irradiated from below.

[0077] Under the above-described environment (conditions), the time spent by the non-human animal 3 in the first space 1 within a predetermined time (for example, 30 minutes) is measured (confirmation step). The same is true for the control group. This test is the so-called OPA (Optogenetic Place Aversion) test.

[0078] If the test results show that the time spent in the first space 1 by the above-mentioned non-human animal 3 that has undergone the test substance administration step is significantly longer than the time spent in the first space 1 by the non-human animals of the control group, the test substance administered to the non-human animal 3 in the test substance administration step can be selected as a candidate drug that reduces the expression level or function of at least one of Nav1.7, 1.8, and 1.9.

[0079] In contrast, if the time spent in the first space 1 by the above-mentioned non-human animal 3 that has undergone the test substance administration step is significantly shorter than the time spent in the first space 1 by the non-human animals of the control group, the test substance administered to the non-human animal 3 in the test substance administration step can be selected as a candidate drug that increases the expression level or enhances the function of at least one of Nav1.7, 1.8, and 1.9.

[0080] For example, if the above-mentioned non-human animal 3 co-expresses only Nav1.7 out of Nav1.7, 1.8 and 1.9, with channelrhodopsin 2, the candidate drug to be selected will be a candidate drug that reduces the expression level or reduces the function of Nav1.7, or a candidate drug that increases the expression level or enhances the function of Nav1.7.

[0081] Furthermore, the ratio of the length of time spent in the first space 1 to the total time spent in the first space 1 and the second space 2 may be calculated from the time spent by the non-human animal in the first space 1, and this ratio may be compared with that of a control group.

[0082] <Pain sensitivity increase process> The drug screening method of this embodiment may further include a step of increasing the pain sensitivity of the non-human animal (pain sensitivity increasing step) prior to the above-mentioned test substance administration step.

[0083] By carrying out the pain sensitivity increasing step, pain hypersensitivity is induced in the non-human animal, and the non-human animal can be used as a neuropathic pain model. Note that the effect of increasing the pain sensitivity of the non-human animal by the pain sensitivity increasing step is reversible, and the pain sensitivity of the non-human animal gradually decreases to its original level over time.

[0084] In the pain sensitivity increasing step, light of a wavelength that opens the light-sensitive cation channel is irradiated for 30 minutes or more, or one or more compounds selected from the group consisting of FK506, streptozotocin, paclitaxel, cisplatin, oxaliplatin, and vincristine are administered. The administration method of these compounds (administration route, dosage, number of administrations, etc.) can be determined in the same manner as in the test substance administration step.

[0085] For example, when administering FK506, 3 mg per kg of body weight may be administered intraperitoneally every day for 7 consecutive days.

[0086] When streptozotocin is administered, it may be administered intraperitoneally at a dose of 7 mg per kg of body weight for several days.

[0087] In addition, when paclitaxel is administered, it may be administered intraperitoneally at a dose of 2 mg per kg of body weight for several days.

[0088] When cisplatin is administered, it may be administered intraperitoneally at a dose of 2 mg per kg of body weight for several days.

[0089] In addition, when oxaliplatin is administered, it may be administered intraperitoneally at an amount of 5 mg per kg of body weight for several days.

[0090] Additionally, when vincristine is administered, it may be administered intraperitoneally at a dose of 0.2 mg per kg of body weight for several days.

[0091] On the other hand, when irradiating for 30 minutes or more with light of a wavelength that opens light-sensitive cation channels, the light intensity can be, for example, 1 to 10 mV, 1.2 to 8 mV, 5 to 8 mV, 1.5 to 2.25 mV, 1 to 3 mV, 1.5 to 10 mV, or 1.2 mV or more. Because pain continues to occur in non-human animals during light irradiation, it is preferable to administer general anesthesia before irradiation. When pain sensitivity is increased by light irradiation, the effect lasts for about 24 to 48 hours, but the higher the light intensity, the longer the period of increased pain sensitivity.

[0092] Whether or not the pain sensitivity of a non-human animal has increased as a result of the pain sensitivity increasing step can be determined, for example, by whether or not the threshold for stimuli that elicit escape behavior is significantly lowered compared to a control group that has not undergone the pain sensitivity increasing step. If the threshold for stimuli that elicit escape behavior is significantly lowered compared to the control group, it is recognized that light sensitivity has increased.

[0093] Whether or not the threshold for stimulation has been significantly lowered can be confirmed, for example, by the so-called von Frey test (a test in which a filament is applied to the sole of the foot to measure the threshold for withdrawal (paw withdrawal)), or by irradiating the sole of the foot with light of different intensities at a wavelength at which light-sensitive cation channels open. If the threshold for stimulation perceived as pain has been lowered, the subject will exhibit withdrawal behavior by withdrawing their paw even with lower intensity light. If the von Frey test or light irradiation is performed after the pain sensitivity increasing step and it is not confirmed that the threshold for stimulation that induces withdrawal behavior has been lowered, it may be possible to increase the intensity of light irradiated in the pain sensitivity increasing step, or to increase the administration period or dosage of one or more of the above-mentioned compounds.

[0094] Even when the pain sensitivity increasing process is carried out, as in the case where it is not carried out, by confirming whether or not the subject escapes from the light (in other words, pain) irradiated in the irradiation process (confirmation process), it is possible to determine whether or not the test substance administered in the test substance administration process has the activity of increasing or decreasing the expression level, or enhancing or decreasing the function, of at least one of Nav1.7, 1.8 and 1.9.

[0095] When the pain sensitivity increasing step has been carried out and the result of the confirmation step shows that the test substance administered in the test substance administration step reduces the expression level or function of at least one of Nav1.7, 1.8, and 1.9, the test substance can also be selected as a candidate substance for a therapeutic agent (ameliorator) for neuropathic pain. In other words, when the drug screening method of this embodiment includes the pain sensitivity increasing step, the drug screening method can also be used as a method for screening a therapeutic agent (ameliorator) for neuropathic pain.

[0096] The drug screening method of this embodiment has been described in detail above. According to this embodiment, the effect of a test substance on the expression and / or function of at least one of Nav1.7, 1.8, and 1.9 can be confirmed non-invasively while keeping the non-human animal alive, thereby making it possible to reduce the burden on the non-human animal compared to conventional screening methods.

[0097] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included in the scope of the present invention. [Example]

[0098] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0099] [Experimental Example 1] In this experiment, using the above-mentioned crossbreeding method shown in Figure 1, we generated mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons, mice expressing Nav1.8 and channelrhodopsin 2 in dorsal root ganglion neurons, and mice expressing Nav1.9 and channelrhodopsin 2 in dorsal root ganglion neurons.

[0100] These mice are homozygous for one allele in which a gene encoding iCre recombinase is inserted downstream of Nav1.x (x = 7-9), and another allele in which a gene encoding channelrhodopsin 2 is inserted downstream of two loxP sequences. The parent Flox mice, which have a stop codon flanked by two loxP sequences downstream of the CAG promoter and further downstream carry the genes for ChR2 and EYFP, were commercially available. The mice carrying the gene encoding iCre recombinase downstream of Nav1.x were generated by the present inventors (see the PLoS One publication mentioned above).

[0101] In the above mice, the gene encoding Nav1.7 consists of the nucleotide sequence shown in SEQ ID NO: 1. The gene encoding Nav1.8 consists of the nucleotide sequence shown in SEQ ID NO: 2. The gene encoding Nav1.9 consists of the nucleotide sequence shown in SEQ ID NO: 3. Furthermore, the gene encoding ChR2 consists of the nucleotide sequence shown in SEQ ID NO: 4. The following experiments were performed using mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons.

[0102] [Experimental Example 2] In this experiment, the mice expressing Nav1.7 and channelrhodopsin 2 in the dorsal root ganglion neurons were intraperitoneally administered 3 mg of FK506 per kg of body weight every day for 7 days (test substance administration step). FK506 is a substance known to induce neuropathic pain.

[0103] Before administration of FK506 and on the 11th day after the start of administration, blue light was irradiated onto the hind paws of the mice a total of six times, and the number of times the mice exhibited escape behavior (paw withdrawal) was counted and the percentage of times they exhibited escape behavior was calculated (plantar irradiation test; see Figure 2 for the irradiation procedure).

[0104] FIG. 4 is a graph showing the relationship between the ratio of the number of times escape behavior occurred to the total number of times light was irradiated and the intensity of blue light in mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons.

[0105] As shown in Figure 4, the percentage of escape behaviors was higher in the Post (11 days after FK506 administration) group than in the control group Pre (before FK506 administration), even at the same blue light intensity. Note that in Figure 4, an asterisk (*) indicates p<0.05.

[0106] This suggests that administration of FK506 increased the expression level or function of Nav1.7, and therefore suggests that FK506 can be selected as a candidate drug that increases the expression level or function of Nav1.7.

[0107] [Experimental Example 3] In this experiment, mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons were intraperitoneally administered FK506 at a dose of 3 mg / kg body weight for 7 consecutive days. After the start of administration, the mice were placed in the environment shown in Figure 3 once every 4 days. Blue light was irradiated onto the hind paws in Space 1 (1) and green light was irradiated onto the hind paws in Space 2 (2). The proportion of the time spent by the mice in Space 1 relative to the total time was calculated (OPA test).

[0108] In addition, a control group was prepared separately to which physiological saline was administered instead of FK506, and similarly subjected to the OPA test.

[0109] FIG. 5 is a graph showing the results of an OPA test in which mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons were administered FK506.

[0110] As shown in Figure 5, the mice administered with FK506 spent significantly less time in the first space 1 irradiated with blue light than the control mice administered with saline at 8 and 12 days after the start of FK506 administration. In Figure 5, an "* (asterisk)" indicates p<0.05.

[0111] These results also suggest that administration of FK506 increased the expression level or function of Nav1.7, suggesting that FK506 can be selected as a candidate drug for increasing the expression level or function of Nav1.7.

[0112] [Experimental Example 4] In this experiment, mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons were administered FK506 at a dose of 3 mg / kg body weight for 7 consecutive days. A control group was also administered saline instead of FK506. The expression levels of Nav1.7 mRNA and β-actin mRNA in the dorsal root ganglion neurons of these mice were then measured by PCR.

[0113] Figure 6 is a graph showing the results of administering FK506 to mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons, and measuring the expression levels of Nav1.7 mRNA and β-actin mRNA in dorsal root ganglion neurons.

[0114] As shown in FIG. 6, 11 days after the start of FK506 administration, the expression level of Nav1.7 mRNA was significantly higher than in the control group.

[0115] These results confirmed that administration of FK506 actually increased the expression level of Nav1.7 in non-human animals.

[0116] [Experimental Example 5] In this experiment, mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons, mice expressing Nav1.8 and channelrhodopsin 2 in dorsal root ganglion neurons, and mice expressing Nav1.9 and channelrhodopsin 2 in dorsal root ganglion neurons (mice produced in Experimental Example 1) were irradiated with blue light for 30 minutes on the soles of one of their hind paws under general anesthesia (pain sensitivity increase process). Thereafter, a filament was pressed against the plantar surface of each mouse that had been irradiated for 30 minutes or the plantar surface of the opposite, unirradiated side, and the threshold for withdrawal behavior was measured (the so-called von Frey test).

[0117] Figure 7 is a graph showing the results of a von Frey test performed on mice expressing Nav1.7 and channelrhodopsin 2 in dorsal root ganglion neurons after a pain sensitivity increase procedure. Figure 8 is a graph showing the results of a von Frey test performed on mice expressing Nav1.8 and channelrhodopsin 2 in dorsal root ganglion neurons after a pain sensitivity increase procedure. Figure 9 is a graph showing the results of a von Frey test performed on mice expressing Nav1.9 and channelrhodopsin 2 in dorsal root ganglion neurons after a pain sensitivity increase procedure. In Figures 7 to 9, an "* (asterisk)" indicates p<0.05 compared to the contralateral, and a "#" indicates p<0.05 compared to before 30 minutes of blue light irradiation.

[0118] As shown in Figures 7 to 9, in all mice co-expressing Nav1.7 and channelrhodopsin 2, mice co-expressing Nav1.8 and channelrhodopsin 2, and mice co-expressing Nav1.9 and channelrhodopsin 2, the pain threshold was temporarily lower in one of the left and right hind paws (ipsilateral) that was exposed to blue light for 30 minutes compared to the other unexposed paw (contralateral).

[0119] These results demonstrate that exposure to light with wavelengths that open light-sensitive cation channels for 30 minutes or more can increase pain sensitivity and induce reversible pain hypersensitivity. Therefore, these mice after 30 minutes or more of exposure are in a state where neuropathic pain has developed, and can be used as an animal model of neuropathic pain. [Industrial Applicability]

[0120] According to the present invention, it is possible to non-invasively screen for drugs that increase or decrease the expression level or enhance or decrease the function of at least one of Nav1.7, 1.8, and 1.9, and therefore the present invention is industrially applicable. [Explanation of symbols]

[0121] 1...first space, 2...second space, 3...non-human animal, 5...glass plate, 6...light irradiation device, 10...container, 10a...floor surface

Claims

1. A method for screening for a drug that increases or decreases the expression level of at least one of voltage-gated sodium channels 1.7 (Nav1.7), 1.8 (Nav1.8), and 1.9 (Nav1.9), or enhances or decreases the function of said at least one, comprising: administering a test substance to a non-human animal that expresses at least one of Nav1.7, 1.8, and 1.9 and a light-sensitive cation channel in dorsal root ganglion neurons; A drug screening method comprising the steps of: irradiating the non-human animal after administration of a test substance with light of a wavelength at which the light-sensitive cation channel opens; and confirming whether the non-human animal escapes from the irradiated light.

2. The drug screening method according to claim 1, further comprising the step of increasing the pain sensitivity of the non-human animal prior to the step of administering the test substance.

3. 3. The drug screening method according to claim 2, wherein the step of increasing the sensitivity involves irradiating the cells with the light for 30 minutes or more.

4. The drug screening method according to claim 2, wherein the step of increasing sensitivity comprises administering to the non-human animal a compound selected from the group consisting of FK506, streptozotocin, paclitaxel, cisplatin, oxaliplatin, and vincristine.

5. The drug screening method according to claim 1, wherein the light-sensitive cation channel is channelrhodopsin 2.

6. The drug screening method according to any one of claims 1 to 5, wherein in the step of confirming whether or not the non-human animal escapes from the light, the ratio of the number of times the non-human animal escapes from the light to the number of times the non-human animal is irradiated with the light is confirmed.

7. The drug screening method of claim 6, wherein if the percentage of times the subject escapes from the light is lower than that of a control group not administered the test substance, the test substance is selected as a candidate drug that reduces the expression level or function of at least one of Nav1.7, 1.8 and 1.

9.

8. The drug screening method of claim 6, wherein if the percentage of times the subject escapes from the light is higher than that of a control group not administered the test substance, the test substance is selected as a candidate drug that increases the expression level or enhances the function of at least one of Nav1.7, 1.8 and 1.

9.

9. The drug screening method according to any one of claims 1 to 5, wherein in the step of confirming whether or not the non-human animal escapes from the light, the time the non-human animal spends in the first space is measured in an environment in which the non-human animal can move freely between a first space to which the light is irradiated and a second space to which the light is not irradiated.

10. The drug screening method of claim 9, wherein the test substance is selected as a candidate drug that reduces the expression level or function of at least one of Nav1.7, 1.8 and 1.9 if the test substance spends a longer time in the first space than a control group that has not been administered the test substance.

11. The drug screening method of claim 9, wherein the test substance is selected as a candidate drug that increases the expression level or enhances the function of at least one of Nav1.7, 1.8 and 1.9 if the time spent in the first space is shorter than that of a control group that has not been administered the test substance.

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