Use of an adenosine A2B receptor as a target for screening, development or manufacture of drugs for the prevention and / or treatment of itching

Abstract

The present invention provides the use of adenosine A2B receptors as targets in medicaments for the prevention and / or treatment of itching and belongs to the field of biopharmaceutical technology. Based on previously reported single-cell RNA sequencing data from the mouse dorsal root ganglion (DRG), the present invention investigates the expression of genes encoding important ligand ion channels and demonstrates high expression of Gfral and Adora2b in neuron_10, suggesting that the receptor for Adora2b, the adenosine A2B receptor, may be involved in the regulation of itching. The results of the itch behavior experiments showed that histamine-induced scratching behavior in mice could be effectively prevented by blocking the activity of the adenosine A2B receptor. This suggests that A2B receptors are involved in the histamine-induced sensation of itching.The present invention also provides a use of a reagent that inhibits the biological function of adenosine A2B for the manufacture of medicaments for the prevention and / or treatment of itching, thereby providing a new agent for the prevention and treatment of itching.

Description

TECHNICAL FIELD

[0001] The present invention belongs to the field of biotechnological drug technology and relates in particular to the use of adenosine A2B receptors as a target in drugs for the prevention and / or treatment of itching. STATE OF THE ART

[0002] Itching is the primary complaint of patients seeking treatment at a dermatology clinic. It is caused by harmful stimuli such as inflammatory mediators, neurotransmitters, and neuropeptides that activate nerve endings in the skin, resulting in itch signals that are transmitted from the peripheral skin, via the spinal cord and thalamus, to the cerebral cortex. Depending on the duration of the itching symptoms, itching can be classified as acute or chronic. Acute itching lasts less than six weeks, while chronic itching lasts longer than six weeks. Chronic itching is widespread and, due to the persistent itching and extensive lesions, is often accompanied by depression and insomnia, significantly impacting patients' quality of life.

[0003] Epidemiological studies have shown that important non-inflammatory conditions such as uremic pruritus are associated with itchy skin in approximately 22% to 90% of patients (Feramisco et al. 2010). Currently, antihistamines remain the mainstay of clinical anti-itch medications, but they have limited efficacy in most cases of chronic itch. Therefore, further analysis of the mechanisms of itch induction and regulation, as well as the identification of effective target molecules, will provide important theoretical insights for the development of novel anti-itch drugs. However, no targets for anti-itch drugs have yet been reported. CONTENT OF THE PRESENT INVENTION

[0004] In view of this, the present invention aims to provide a use of the adenosine A2B receptor as a target for the screening, development or manufacture of drugs for the prevention and / or treatment of itching.

[0005] The present invention provides a use of the adenosine A2B receptor as a target for the screening, development or manufacture of medicaments for the prevention and / or treatment of itching.

[0006] Preferably, the itching includes at least one of the following: uremic pruritus, chronic pruritus, and acute pruritus.

[0007] Preferably, the itching is triggered by histamine or chloroquine.

[0008] Preferably, the drug comprises a component that inhibits the activity of the adenosine A2B receptor.

[0009] The present invention provides a use of a reagent that inhibits the biological function of adenosine A2B for the manufacture of medicaments for the prevention and / or treatment of itching.

[0010] Preferably, the reagent comprises a monoclonal or polyclonal antibody against the adenosine A2B receptor, an antagonist of the adenosine A2B receptor, and a competitive ligand for the adenosine A2B receptor.

[0011] Preferably, the adenosine A2B receptor antagonist comprises PSB-603.

[0012] Preferably, PSB-603 is administered at a concentration of 10 µM or higher.

[0013] Preferably, the itching includes at least one of the following: uremic pruritus, chronic pruritus, and acute pruritus.

[0014] Preferably, the itching is triggered by histamine or chloroquine.

[0015] The present invention provides a use of the adenosine A2B receptor as a target for the screening, development, or manufacture of medicaments for the prevention and / or treatment of itching. Based on previously reported single-cell RNA sequencing data from the mouse dorsal root ganglion (DRG), the present invention investigates the expression of genes encoding important ligand ion channels and demonstrates high expression of Gfral and Adora2b in neuron_10, suggesting that the receptor for Adora2b, the adenosine A2B receptor, may be involved in the regulation of itching. The results of itch behavior experiments showed that histamine-induced scratching behavior in mice could be effectively prevented by blocking the activity of the adenosine A2B receptor. This suggests that A2B receptors are involved in the histamine-induced sensation of itching.Therefore, adenosine A2B receptors, as target molecules for drugs, are an effective means of treating itching. BRIEF DESCRIPTION OF THE DRAWINGS Fig. Figure 1 shows the results of the classification of DRG neurons based on gene expression profiles, where A represents the Umap-embedded projection of DRG neuron subtypes in adult mice. This number indicates the number of seurat clusters at a resolution of 0.4. Figure B is a dot plot showing the expression profiles of the first three characteristic differentially expressed genes (fDEGs) in the DRG neuron subtype. The dot size indicates the percentage of positive cells, and the color intensity indicates the average expression of the gene in question. Figure C is a dot plot of the cell type marker expression profiles. Figure D is a summary based on previous neuronal classifications. Fig. Figure 2 shows a dot plot of gene expression encoding ATP, adenosine, and cannabinoid receptors. Note: The dot size indicates the percentage of gene-positive cells, and the color intensity indicates the average expression of the gene in question. Cell types were analyzed based on the percentage and average expression of relevant genes in clusters. D2 approach. Fig. Figure 3 shows the results of the involvement of A2B receptors in the regulation of itch, and A is a representative image of A2B-positive neurons in the DRG and the labeling of GFRα1 (arrowheads). Scale bar, 20 µm. B is the proportion of A2B-positive cells co-expressing GFRα-1 in the DRG (n = 3 mice). C is intrathecal injection of PSB-603 for dose-dependent inhibition of histamine-induced scratching behavior (histamine: His; PSB-603: P; n = 7 to 8 mice per group; data are presented as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001, one-sided ANOVA followed by Tukey's multiple comparison test). Fig. Figure 4 shows the results of the inhibition of chloroquine-induced scratching behavior by PSB 603 in a dose-dependent manner. DETAILED DESCRIPTION

[0016] The present invention provides a use of the adenosine A2B receptor as a target for the screening, development or manufacture of medicaments for the prevention and / or treatment of itching.

[0017] The present invention is not specifically limited to this type of itching, and itching caused by harmful stimuli such as inflammatory mediators, neurotransmitters, and neuropeptides falls within the category of itching to which the present invention relates, e.g., uremic pruritus, chronic pruritus, and acute pruritus. Within the scope of the present invention, the itching is preferably histamine- or chloroquine-induced itching. It has been demonstrated that epidermal injections of histamine or chloroquine effectively induce scratching behavior in mice.

[0018] In the present invention, the drug achieves its therapeutic purpose preferably by inhibiting the activity of the adenosine A2B receptor. Single-cell transcriptome data were downloaded from the Gene Expression Omnibus (GEO) database (http: / / www.ncbi.nlm.nih.gov / geo / ) SE155622. (Wang, K., Wang, S., Chen, Y., Wu, D., Hu, Tochitsky, I., Yang, L., Cheng, Y.-C., Li, E., Kawaguchi, R., et al. (2020a). Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after Axonal Injury. j.neuron.2020.07.026). scRNA data were downloaded from the UCSC website (https: / / cells.ucsc.edu / ?ds=mouse-nervous-system+drg) (Speir, M L., Bhaduri, A., Markov, N .S., Moreno, P., Nowakowski, TJ, Papatheodorou, I., et al.(2021). UCSC Cell Browser: Visualize Your Single-CellData. Bioinformatics 37, 4578-4580). Analysis of single-cell RNA sequencing data from the mouse DRG revealed high expression of Gfral and Adora2b in neuron_10, suggesting that the Adora2b gene and the A2B receptor it encodes may also be involved in regulating itch. Based on this, immunohistochemical experiments were performed within the scope of the present invention to detect the co-expression of the A2B receptor and GFRα-1 protein in DRG neurons. Itching behavior experiments, designed to investigate whether A2B receptors are involved in the regulation of itching behavior, showed that A2B receptor activity was blocked by intrathecal injection of a selective A2B receptor antagonist, PSB-603, and that administration of PSB-603 effectively prevented histamine-induced scratching behavior.This suggests that inhibiting adenosine A2B receptors is an effective means of treating itching.

[0019] The present invention provides a use of a reagent that inhibits the biological function of adenosine A2B for the manufacture of medicaments for the prevention and / or treatment of itching.

[0020] In the present invention, the reagent preferably comprises a monoclonal or polyclonal antibody against the adenosine A2B receptor, an adenosine A2B receptor antagonist, and a competitive ligand for the adenosine A2B receptor. In the present embodiment of the invention, the pharmacological efficacy against itching is demonstrated using the example of an adenosine A2B receptor antagonist. The adenosine A2B receptor antagonist preferably comprises PSB-603.

[0021] It has been shown that the reduction in the animals' scratching behavior is related to the dosage concentration of PSB-603, and that PSB-603 is preferably administered at a concentration of 10 µM or more, and that the number of scratches in the animals cannot be reduced when it is administered at a concentration of 1 µM.

[0022] The application of an adenosine A2B receptor provided by the present invention as a target in a medicament for the prevention and / or treatment of itching is described in detail below in conjunction with the exemplary embodiments; however, they are not to be understood as limiting the scope of protection of the present invention. Example 1: Screening of a gene that regulates the sensation of itching

[0023] Based on published data from single-cell RNA sequencing of the mouse DRG (Wang et al., 2021; Renthal et al., 2020; Speir et al., 2021), it was found that gene expression in different types of DRG neurons is related to their morphological and functional characteristics (Li et al., 2016). As a result, it was found that neuron 10 expresses high levels of Mrgpra3 (Mas-associated G protein-coupled receptor A3), which may be involved in the regulation of itching (Li et al., 2016; Speirs and P. Seal, 2016). Fig. 1).

[0024] Based on previous studies, the expression of genes encoding important ligand ion channels was analyzed. Ion channel-related genes were obtained from the org. Mm. eg .db (v 3 .13 .0) R package, and the SeuratR package was used to examine the expression of these genes in the mouse DRG single-cell RNA sequencing database. In Neuron_10, Gfral and Adora2b were highly expressed ( Fig. 2) ATP and adenosine receptors are purinergic receptors involved in modulating nociception (Burnstock, 2016; Inoue, 2022). The receptors encoded by the Adora2b gene are all purinergic receptors. The single-cell sequencing data mentioned above suggest that neuron 10 may be involved in regulating itch sensation. Therefore, the Adora2b gene, which is highly expressed in neuron 10, and the A2B receptor it encodes could also be involved in regulating itch. Example 2

[0025] Expression of A2B receptors in DRG neurons, demonstrated by immunohistochemical experiments

[0026] The steps of immunohistochemistry are as follows: After intraperitoneal anesthesia with 1% sodium pentobarbital, the mice underwent cardiac perfusion with 0.1 M PBS and 4% PFA, followed by spinal cord isolation and harvesting of DRG samples (lumbar segments 4-5). Isolated DRG tissue was refixed with 4% PFA solution for 12 hours and then dehydrated with 15% and 30% sucrose solutions, respectively. After dehydration, the sections were embedded using OCT and cryosected to a thickness of 15 µM and mounted on slides. The DRG slides were then washed three times for 10 minutes each with PBS solution. They were subsequently incubated for one hour at room temperature with the prepared containment buffer (PBS solution with 5% goat serum and 0.5% TritonX-100).Primary antibodies (rabbit anti-A2B, 1:100; mouse anti-GFRα-1, 1:100) were applied with antibody dilutions (2.5% goat serum and 0.05% Triton X-100 in PBS solution), followed by overnight incubation at 4°C. The next day, the slides were washed five times for 10 minutes each with PBS. The appropriate fluorescent secondary antibody (donkey anti-mouse, donkey anti-rabbit, both 1:500) was selected according to the type of primary antibody. The slides were then placed in a dark, light-protected box and incubated for 2 hours at room temperature. After incubation with the secondary antibodies, the slides were washed three times for 10 minutes each with PBS solution. After drying in a light-protected environment, the slides were sealed with a DAPI-containing, fluorescent-inhibiting sealer.After the sealant had hardened, the fluorescence reaction of the DRG sections was observed and photographed using confocal microscopy.

[0027] The results are in Fig. Figure 3 shows that A2B receptor and GFRα1 proteins were co-expressed in DRG neurons (A in Fig. 3), and approximately 69% of the A2B-positive neurons overlapped with GFRα 1 (B in Fig. 3). Example 3

[0028] Experimental study on itch behavior to investigate whether A2B receptors in the DRG are involved in the regulation of itch behavior.

[0029] See the state of the art (Jang et al., 2015) for itch behavior research, as described: Two days prior to the experiment, mice were shaved at the necks and placed in transparent bioplastic boxes for at least one hour per day for two consecutive days to acclimatize. Adjacent transparent plastic boxes were separated by sheets of white paper to prevent mutual interference between mice. On the day of the experiment, the mice received a subcutaneous injection of histamine (250 µg / 50 µl) into the neck and were immediately placed in a transparent plastic box, where a video camera recorded scratching behavior over a period of 30 minutes. A valid scratch was defined as a mouse lifting its hind foot, scratching the skin at the histamine injection site, and then lowering its hind foot again.

[0030] A single valid scratching behavior was assumed in mice between the lifting and lowering of their hind foot, regardless of how often they scratched. To investigate the antipruritic effect of PSB-603, mice were administered PSB-603 (dose 10 µl, concentration 1 µM or 10 µM) intrathecally 30 minutes before histamine injection.

[0031] The results showed that injection of histamine into the epidermis of normal mice effectively induced scratching behavior. A pharmacological approach was used to block A2B receptor activity by intrathecal injection of the selective A2B receptor antagonist PSB-603. Prior administration of PSB-603 effectively blocked histamine-induced scratching behavior. Furthermore, the attenuating effect of PSB-603 on scratching was dose-dependent. PSB-603 reduced the number of histamine-induced scratches at a dose of 10 µM, but not at 1 µM (Cmax in 10 µM). Fig. 3) These results suggest that A2B receptors are involved in the histamine-induced sensation of itching. Therefore, inhibiting adenosine A2B receptors is an effective method for treating itching. Example 4

[0032] Experimental study on itch behavior to investigate whether A2B receptors in the DRG are involved in the regulation of itch behavior.

[0033] See the state of the art (Jang et al., 2015) for itch behavior research, as described: Two days prior to the experiment, mice were shaved at the necks and placed in transparent bioplastic boxes for at least one hour per day for two consecutive days to acclimatize. Adjacent transparent plastic boxes were separated by sheets of white paper to prevent mutual interference between mice. On the day of the experiment, the mice received a subcutaneous injection of chloroquine (200 µg / 50 µl) into the neck and were immediately placed in a transparent plastic box, where a video camera recorded scratching behavior over a period of 30 minutes. A valid scratch was defined as a mouse lifting its hind foot, scratching the skin at the chloroquine injection site, and then lowering its hind foot again.A single valid scratching behavior was assumed in mice between the lifting and lowering of their hind foot, regardless of how often they scratched. To investigate the antipruritic effect of PSB-603, mice were administered PSB-603 intrathecally (dose 10 µl, concentration 1 µM, 10 µM, or 20 µM) 30 minutes before chloroquine injection.

[0034] The results showed that injection of chloroquine into the epidermis of normal mice effectively induced scratching behavior. A pharmacological approach was used to block A2B receptor activity by intrathecal injection of the selective A2B receptor antagonist PSB-603. Prior administration of PSB-603 effectively blocked chloroquine-induced scratching behavior. Furthermore, the attenuating effect of PSB-603 on scratching was dose-dependent. PSB-603 reduced the number of chloroquine-induced scratches at doses of 10 µM and 20 µM, but not at 1 µM. Fig. 4) These results suggest that A2B receptors are involved in the chloroquine-induced itching sensation. Therefore, inhibition of adenosine A2B receptors is an effective method for treating itching. literature Burnstock, G. (2016). Purinergic Mechanisms and Pain. Adv. Pharmacol. 75, 91-137. doi: 10.1016 / bs.apha.2015.09.001. Inoue, K. (2022). The Role of ATP Receptors in Pain Signaling. Neurochem. Res. 47, 2454-2468. doi: 10.1007 / s11064-021-03516-6. Jang, Y., Lee, W.-J., Hong, G.-S., and Shim, W.-S. (2015). Red ginseng extract blocks histamine-dependent itch by inhibition of H1R / TRPV1 pathway in sensory neurons. J. Ginseng Res. 39, 257-264. doi: 10.1016 / j.jgr.2015.01.004 Li, C. L., Li, K. C., Wu, D., Chen, Y., Luo, H., Zhao, J. R., et al. (2016). Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity. Cell Res. 26. doi: 10.1038 / cr.2015.149. Feramisco, J.D., T.G. Berger, and M. Steinhoff (2010). Innovative management of pruritus. Dermatol Clin. 28(3): p. 467-78.Peirs, C., and P. Seal, R. (2016). Neural circuits of pain: Recent advances and current perspectives. Science (80-.). 354, 578-583. Renthal, W., Tochitsky, I., Yang, L., Cheng, Y.-C., Li, E., Kawaguchi, R., et al. (2020). Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after Axonal Injury. Neuron 108, 128-144.e9. doi: 10.1016 / j.neuron.2020.07.026. Speir, M. L., Bhaduri, A., Markov, N. S., Moreno, P., Nowakowski, T. J., Papatheodorou, I., et al. (2021). UCSC Cell Browser: Visualize Your Single-Cell Data. Bioinformatics 37, 4578-4580.doi: 10.1093 / bioinformatics / btab503. Wang, K., Wang, S., Chen, Y., Wu, D., Hu, X., Lu, Y., et al. (2021). Single-cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic pain. Cell Res. 31. doi: 10.1038 / s41422-021-00479-9.

[0035] The foregoing are only preferred embodiments of the present invention, and it should be noted that a person with ordinary technical knowledge may make a number of improvements and embodiments without departing from the principles of the present invention, and such improvements and embodiments should be considered to be within the scope of protection of the present invention. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited non-patent literature

[0000] Feramisco, et al. 2010

[0003] Wang, K., Wang, S., Chen, Y., Wu, D., Hu, X., Lu, Y., et al. (2021). Single cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic pain. Cell Res.31.doi:10.1038 / s41422 021 00479 9

[0018] Renthal, W., Tochitsky, I., Yang, L., Cheng, Y.-C., Li, E., Kawaguchi, R., et al.(2020a). Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after Axonal Injury. Neuron 108, 128-144.e9. doi: 10 .10 16 / j.neuron.2020.07.026

[0018] Speir, M L., Bhaduri, A., Markov,N .S., Moreno, P.,Nowakowski,T.J., Papatheodorou,I., et al. (2021). UCSC Cell Browser:Visualize Your Single-CellData .Bioinformatics 37,4578 -4580

[0018] Wang et al. ,2021; Renthal etal., 2020; Speir et al.,2021

[0023] Li et al., 2016

[0023] Li et al., 2016; Peirs und P. Seal, 2016

[0023] Burnstock, G. (2016). Purinergic Mechanisms and Pain. Adv. Pharmacol. 75, 91-137. doi: 10.1016 / bs.apha.2015.09.001

[0034] Inoue, K. (2022). The Role of ATP Receptors in Pain Signaling. Neurochem. Res. 47, 2454-2468. doi: 10.1007 / s11064-021-03516-6

[0034] Jang, Y., Lee, W.-J., Hong, G.-S., and Shim, W.-S. (2015). Red ginseng extract blocks histamine-dependent itch by inhibition of H1R / TRPV1 pathway in sensory neurons. J

[0034] Ginseng Res. 39, 257-264. doi: 10.1016 / j.jgr.2015.01.004

[0034] Li, C. L., Li, K. C., Wu, D., Chen, Y., Luo, H., Zhao, J. R., et al. (2016). Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity. Cell Res. 26. doi: 10.1038 / cr.2015.149

[0034] Feramisco, J.D., T.G. Berger, and M. Steinhoff (2010). Innovative management of pruritus. Dermatol Clin. 28(3): p. 467-78.Peirs, C., and P. Seal, R. (2016). Neural circuits of pain: Recent advances and current perspectives. Science (80-.). 354, 578-583

[0034] Renthal, W., Tochitsky, I., Yang, L., Cheng, Y.-C., Li, E., Kawaguchi, R., et al. (2020)

[0034] Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after

[0034] Axonal Injury. Neuron 108, 128-144.e9. doi: 10.1016 / j.neuron.2020.07.026

[0034] Speir, M. L., Bhaduri, A., Markov, N. S., Moreno, P., Nowakowski, T. J., Papatheodorou, I., et al. (2021). UCSC Cell Browser: Visualize Your Single-Cell Data. Bioinformatics 37, 4578-4580.doi: 10.1093 / bioinformatics / btab503

[0034] Wang, K., Wang, S., Chen, Y., Wu, D., Hu, X., Lu, Y., et al. (2021). Single-cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic pain. Cell Res. 31. doi: 10.1038 / s41422-021-00479-9

[0034]

Claims

[1] Use of adenosine A2B receptor as a target for the screening, development or manufacture of drugs for the prevention and / or treatment of itching. [2] Use according to claim 1, characterized by that the itching includes at least one of the following: uremic pruritus, chronic pruritus and acute pruritus. [3] Use according to claim 1, characterized by that the itching is caused by histamine or chloroquine. [4] Use according to claim 1, characterized by that the medicinal product contains a component that inhibits the activity of the adenosine A2B receptor. [5] Use of a reagent that inhibits the biological function of adenosine A2B for the manufacture of medicaments for the prevention and / or treatment of itching. [6] Use according to claim 5, characterized bythat the reagent comprises a monoclonal or polyclonal antibody against the adenosine A2B receptor, an antagonist of the adenosine A2B receptor, and a competitive ligand for the adenosine A2B receptor. [7] Use according to claim 6, characterized by that the adenosine A2B receptor antagonist includes PSB-603. [8] Use according to claim 7, characterized by that PSB-603 is administered at a concentration of 10 µM or more. [9] Use according to claim 5, characterized by that the itching includes at least one of the following: uremic pruritus, chronic pruritus and acute pruritus. [10] Use according to claim 5, characterized by that the itching is caused by histamine or chloroquine.

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