Use of adenosine a2b receptor as a target in a medicament for preventing and / or treating pruritus

Abstract

The application provides application of an adenosine A2B receptor as a target in a medicine for preventing and / or treating itch, and belongs to the technical field of biological medicines. Based on previously reported single-cell RNA sequencing data of a mouse dorsal root ganglion (DRG), expression of genes encoding main ligand ion channels is detected, Adora2b highly expressed in Neuron_10 is detected, and it is indicated that Adora2b and a receptor (adenosine A2B receptor) of Adora2b can participate in regulating itch. Experiments prove that the activity of the adenosine A2B receptor can be effectively blocked to prevent a histamine-induced scratching behavior of a mouse, and it is indicated that the A2B receptor participates in histamine-induced itch sensation. The application also provides application of a reagent for inhibiting biological functions of the adenosine A2B receptor in preparation of a medicine for preventing and / or treating itch, and provides a new means for prevention and treatment of itch.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and pharmaceutical technology, specifically relating to the application of adenosine A2B receptor as a target in drugs for the prevention and / or treatment of pruritus. Background Technology

[0002] Itching is a common complaint among patients visiting dermatology clinics. It occurs when harmful stimuli such as inflammatory mediators, neurotransmitters, and neuropeptides activate nerve endings in the skin, causing itching signals to travel from the peripheral skin through the spinal cord and thalamus to the cerebral cortex. Based on the duration of the itching symptoms, itching can be classified as acute or chronic. Acute itching lasts less than 6 weeks, while chronic itching lasts longer than 6 weeks. Chronic itching has a higher prevalence in the population. Due to persistent itching and widespread skin lesions, patients often experience complications such as depression and insomnia, leading to a significant reduction in their quality of life.

[0003] Epidemiological surveys have found that in major non-inflammatory diseases, such as uremic pruritus, approximately 22%–90% of patients also experience pruritus (Feramisco, et al., 2010). Currently, antihistamines remain the primary medications used clinically to relieve itching; however, these drugs have limited efficacy for most chronic pruritus cases. Therefore, further elucidation of the mechanisms of itch generation and regulation, as well as effective drug targets, will provide important theoretical guidance for the development of new antipruritic drugs. However, there are currently no reports on drug targets for pruritus. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an adenosine A2B receptor as a target for use in a medicament for the prevention and / or treatment of pruritus.

[0005] This invention provides the application of adenosine A2B receptor as a target in the screening, development, or preparation of drugs for the prevention and / or treatment of pruritus.

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

[0007] Preferably, the itching is histamine- or chloroquine-induced.

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

[0009] This invention provides an agent that inhibits the biological function of adenosine A2B receptor for use in the preparation of medicaments for the prevention and / or treatment of pruritus.

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

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

[0012] Preferably, the dosage concentration of PSB-603 is 10 μM or higher.

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

[0014] Preferably, the itching is histamine- or chloroquine-induced.

[0015] This invention provides the application of adenosine A2B receptor as a target in screening, developing, or preparing drugs for the prevention and / or treatment of pruritus. Based on previously reported single-cell RNA sequencing data from mouse dorsal root ganglia (DRG), this invention detected the expression of genes encoding major ligand ion channels, revealing high expression of Gfra1 and Adora2b in Neuron_10, suggesting that the Adora2b receptor—adenosine A2B receptor—may be involved in regulating pruritus. Pruritus behavior experiments showed that blocking the activity of adenosine A2B receptor effectively prevented histamine-induced scratching behavior in mice. This indicates that the A2B receptor is involved in histamine-induced pruritus sensation. Therefore, adenosine A2B receptor as a drug target is an effective approach for treating pruritus. Attached Figure Description

[0016] Figure 1 The results of DRG neuron classification based on gene expression profiles are shown below. A represents the Umap embedding projection of adult mouse DRG neuron subtypes; the number indicates the number of Seurat clusters at a resolution of 0.4. B is a dot plot showing the expression profiles of the top three characteristic differentially expressed genes (fDEGs) in the DRG neuron subtypes; dot size represents the percentage of positive cells, and color intensity represents the average expression of the relevant genes. C is a dot plot of cell type marker expression profiles. D is an overview summary based on previously reported neuron classification.

[0017] Figure 2 A dot plot of gene expression encoding ATP, adenosine, and cannabinoid receptors; Note: Dot size represents the percentage of gene-positive cells, and color intensity represents the average expression of the relevant gene; Cell types were clustered based on the percentage and average expression of the relevant gene; D2 method;

[0018] Figure 3To illustrate the role of A2B receptors in regulating pruritus, A represents a representative image of A2B-positive neurons and GFRα-1 markers (arrows) in the DRG; scale bar, 20 μm; B represents the proportion of A2B-positive cells expressing GFRα-1 in the DRG (n = 3 mice); C represents the dose-dependent inhibition of histamine-induced scratching behavior by intrathecal injection of PSB-603 (histamine: His; PSB-603: P; n = 7–8 mice per group; data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey multiple comparison test).

[0019] Figure 4 The results show that PSB-603 inhibits chloroquine-induced scratching behavior in a dose-dependent manner. Detailed Implementation

[0020] This invention provides the application of adenosine A2B receptor as a target in the screening, development, or preparation of drugs for the prevention and / or treatment of pruritus.

[0021] This invention does not specifically limit the type of itching described. Itching caused by harmful stimuli such as inflammatory mediators, neurotransmitters, and neuropeptides falls within the scope of this invention, including uremic pruritus, chronic pruritus, and acute pruritus. In this invention, the itching is preferably histamine- or chloroquine-induced. Experiments have shown that epidermal injection of histamine or chloroquine can effectively induce scratching behavior in mice.

[0022] In this invention, the drug preferably achieves its therapeutic purpose by inhibiting the activity of adenosine A2B receptors. Single-cell transcriptomic data were downloaded from the GeneExpression Omnibus (GEO) database (http: / / www.ncbi.nlm.nih.gov / geo / ) for GSE155622 (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) and GSE154659 (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:10.1016 / j.neuron.2020.07.026), scRNA data (Speir, ML, Bhaduri, A., Markov, NS, Moreno, P., Nowakowski, TJ, Papatheodorou, I., et al. (2021). UCSC Cell Browser: Visualize Your Single-Cell Data. Bioinformatics 37, 4578–4580) were downloaded from the UCSC website (https: / / cells.ucsc.edu / ?ds=mouse-nervous-system+drg). Analysis of mouse DRG single-cell RNA sequencing data revealed high expression of Gfra1 and Adora2b in Neuron_10, suggesting that the Adora2b gene and its encoded receptor A2B may also be involved in regulating pruritus. Based on this, the present invention conducted immunohistochemical experiments and found that A2B receptor and GFRα-1 protein were co-expressed in DRG neurons.Experiments on pruritus behavior verified whether A2B receptors are involved in regulating pruritus behavior. Results showed that intrathecal injection of the selective A2B receptor antagonist PSB-603 blocked A2B receptor activity, and administration of PSB-603 effectively prevented histamine-induced scratching behavior. This indicates that inhibiting adenosine A2B receptors is an effective approach to treating pruritus.

[0023] This invention provides an agent that inhibits the biological function of adenosine A2B receptor for use in the preparation of medicaments for the prevention and / or treatment of pruritus.

[0024] In this invention, the reagents preferably include monoclonal or polyclonal antibodies against adenosine A2B receptors, antagonists of adenosine A2B receptors, and competitive ligands of adenosine A2B receptors. In the embodiments of this invention, the efficacy of adenosine A2B receptor antagonists in preventing and treating pruritus is illustrated using them as an example. The preferred adenosine A2B receptor antagonist is PSB-603.

[0025] Experiments have shown that the reduction in scratching behavior in animals is related to the dosage and concentration of PSB-603, and the preferred dosage concentration of PSB-603 is 10 μM or higher. A dosage concentration of 1 μM does not reduce the number of scratches in animals.

[0026] The following detailed description, in conjunction with embodiments, illustrates the application of an adenosine A2B receptor as a target in a drug for the prevention and / or treatment of pruritus provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] Screening for a gene that regulates the sensation of itching

[0029] Using published mouse DRG single-cell RNA sequencing data (Wang et al., 2021; Renthal et al., 2020; Speir et al., 2021), gene expression in different types of DRG neurons was identified as being related to their morphological and functional characteristics (Li et al., 2016). The results showed that Neuron-10 neurons highly expressed Mrgpra3 (Mas-associated G protein-coupled receptor A3), which may be involved in regulating itch sensation (Li et al., 2016; Peirs and P. Seal, 2016). Figure 1 ).

[0030] Building upon prior research, the applicant analyzed the expression of genes encoding major ligand ion channels. Ion channel-related genes were obtained from the org.Mm.eg.db (v 3.13.0) R package, and their expression in the mouse DRG single-cell RNA sequencing database was detected using the Seurat R package. High expression of Gfra1 and Adora2b was detected in Neuron_10. Figure 2 ATP and adenosine receptors are purinergic receptors and are involved in regulating pain sensation (Burnstock, 2016; Inoue, 2022). The receptors encoded by the Adora2b gene are all purinergic receptors. Importantly, the single-cell sequencing data mentioned above indicate that Neuron 10 may be involved in regulating itch sensation. Therefore, the highly expressed Adora2b gene in Neuron 10 and its encoded receptor A2B may also be involved in regulating itch.

[0031] Example 2

[0032] Immunohistochemical assays were used to detect the expression of A2B receptors in DRG neurons.

[0033] The steps of immunohistochemistry are as follows:

[0034] Mice were anesthetized intraperitoneally with sodium pentobarbital (1%) and then perfused with the heart successively using 0.1M PBS and 4% PFA. The spinal cord was then isolated, and DRG tissue samples were collected from the lumbar 4-5 segments. The isolated DRG tissue was post-fixed with 4% PFA solution for 12 hours, followed by dehydration with 15% and 30% sucrose solutions. After dehydration, the tissue was embedded in OCT and frozen sectioned to a thickness of 15 μM, then mounted on adhesive slides. The DRG slides were then washed three times with PBS solution for 10 min each time. Next, they were incubated at room temperature for 1 hour with a prepared blocking buffer (PBS solution containing 5% goat serum and 0.5% Triton X-100). Then, apply the primary antibodies (rabbit anti-A2B, 1:100; mouse anti-GFRα-1, 1:100) to the slides. The antibody dilution solution is PBS solution of 2.5% goat serum and 0.05% Triton X-100. Incubate overnight at 4°C. The next day, wash the slides five times with PBS for 10 minutes each time. Select the appropriate fluorescent secondary antibody (donkey anti-mouse, donkey anti-rabbit, both 1:500) according to the species of the primary antibody. Then place the slides in a dark box and incubate at room temperature for 2 hours. After the secondary antibody incubation, wash the slides three times with PBS solution for 10 minutes each time. After air-drying in the dark, mount the slides with anti-fluorescence quenching mounting medium containing DAPI. After the mounting medium solidifies, observe the fluorescence reaction of the DRG sections using a confocal microscope and take photographs.

[0035] See results Figure 3 A2B receptor and GFRα-1 protein are co-expressed in DRG neurons. Figure 3 In the middle A), and approximately 69% of A2B positive neurons overlap with GFRα-1 ( Figure 3 (B)

[0036] Example 3

[0037] Experimental study on pruritus behavior: Does the A2B receptor in the DRG participate in regulating pruritus behavior?

[0038] The pruritus behavior was described in existing technology (Jang et al., 2015). The specific procedure was as follows: Two days prior to the experiment, the neck fur of mice was shaved, and they were placed in transparent organic plastic boxes for two consecutive days of acclimatization training, at least one hour each day. Adjacent transparent plastic boxes were separated by white paper to prevent interference between mice. On the day of the experiment, mice received a subcutaneous injection of histamine (250 μg / 50 μl) into their necks and were immediately placed in the transparent plastic boxes. Scratching behavior was recorded using a camera over 30 minutes. A valid scratch was defined as the mouse raising its hind paw, scratching the skin at the histamine injection site, and then lowering its hind paw. The number of scratches between raising and lowering the hind paw was considered a valid scratch response. To evaluate the antipruritic effect of PSB-603, mice were administered PSB-603 intrathecally within 30 minutes before histamine injection (dose 10 μl, concentration 1 μM or 10 μM).

[0039] The results showed that epidermal injection of histamine in normal animals effectively induced scratching behavior in mice. Pharmacological methods were employed to block A2B receptor activity through intrathecal injection of the selective A2B receptor antagonist PSB-603. Pre-administration of PSB-603 effectively prevented histamine-induced scratching behavior. Furthermore, the scratch-reducing effect of PSB-603 was dose-dependent. PSB-603 at a dose of 10 μM reduced the number of histamine-induced scratches, while at 1 μM it did not reduce them. Figure 3 (C). These results indicate that the A2B receptor is involved in histamine-induced pruritus. Therefore, inhibiting the adenosine A2B receptor is an effective approach to treating pruritus.

[0040] Example 4

[0041] Experimental study on pruritus behavior: Does the A2B receptor in the DRG participate in regulating pruritus behavior?

[0042] The pruritus behavior was described in existing technology (Jang et al., 2015). The specific procedure was as follows: Two days prior to the experiment, the neck fur of mice was shaved, and they were placed in transparent organic plastic boxes for two consecutive days of acclimatization training, at least one hour each day. Adjacent transparent plastic boxes were separated by white paper to prevent interference between mice. On the day of the experiment, mice received a subcutaneous injection of chloroquine (200 μg / 50 μl) in their necks and were immediately placed in the transparent plastic boxes. Scratching behavior was recorded using a camera over 30 minutes. A valid scratch was defined as the mouse raising its hind paw, scratching the skin at the histamine injection site, and then lowering its hind paw. The number of scratches between raising and lowering the hind paw was considered a valid scratch response. To evaluate the antipruritic effect of PSB-603, mice were administered PSB-603 intrathecally within 30 minutes before chloroquine injection (dose 10 μl, concentrations of 1 μM, 10 μM, or 20 μM).

[0043] The results showed that epidermal injection of chloroquine in normal animals effectively induced scratching behavior in mice. Pharmacological methods were employed to block A2B receptor activity through intrathecal injection of the selective A2B receptor antagonist PSB-603. Pre-administration of PSB-603 effectively prevented chloroquine-induced scratching behavior. Furthermore, the scratch-reducing effect of PSB-603 was dose-dependent. PSB-603 reduced the number of scratches induced by chloroquine at 10 μM and 20 μM doses, but did not reduce it at 1 μM. Figure 4 These results indicate that A2B receptors are also involved in chloroquine-induced itching. Therefore, inhibiting adenosine A2B receptors is an effective approach to treating pruritus.

[0044] References

[0045] Burnstock,G.(2016).Purinergic Mechanisms and Pain.Adv.Pharmacol.75,91–137.doi:10.1016 / bs.apha.2015.09.001.

[0046] Inoue,K.(2022).The Role ofATP Receptors in PainSignaling.Neurochem.Res.47,2454–2468.doi:10.1007 / s11064-021-03516-6.

[0047] Jang,Y.,Lee,W.-J.,Hong,G.-S.,and Shim,W.-S.(2015).Red ginsengextractblocks

[0048] histamine-dependent itch by inhibition of H1R / TRPV1 pathway insensoryneurons.J.

[0049] Ginseng Res.39,257–264.doi:10.1016 / j.jgr.2015.01.004

[0050] 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-cellRNA-sequencing and functional heterogeneity.Cell Res.26.doi:10.1038 / cr.2015.149.

[0051] Feramisco,J.D.,T.G.Berger,and M.Steinhoff(2010).Innovativemanagementofpruritus.Dermatol Clin.28(3):p.467-78.Peirs,C.,and P.Seal,R.(2016).Neuralcircuits of pain:Recent advances and current perspectives.Science(80-.).354,578–583.

[0052] Renthal,W.,Tochitsky,I.,Yang,L.,Cheng,Y.-C.,Li,E.,Kawaguchi,R.,etal.(2020).

[0053] Transcriptional Reprogramming of Distinct Peripheral SensoryNeuronSubtypes after Axonal Injury.Neuron 108,128-144.e9.doi:10.1016 / j.neuron.2020.07.026.

[0054] Speir, ML, Bhaduri, A., Markov, NS, Moreno, P., Nowakowski, TJ, Papatheodorou, I., et al. (2021). UCSC Cell Browser: Visualize Your Single-CellData. Bioinformatics 37, 4578–4580. doi:10.1093 / bioinformatics / btab503.

[0055] 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.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of adenosine A2B receptor as a target in the screening, development, or preparation of drugs for the prevention and / or treatment of pruritus; The drug contains an ingredient that inhibits the activity of adenosine A2B receptors; The adenosine A2B receptor antagonist is PSB-603.

2. Use according to claim 1, characterized in that, The pruritus includes at least one of the following: uremic pruritus, chronic pruritus, and acute pruritus.

3. Use according to claim 1, characterized in that, The itching is histamine- or chloroquine-induced.

4. The use of an agent that inhibits the biological function of adenosine A2B receptor in the preparation of a medicament for the prevention and / or treatment of pruritus, wherein the agent is an antagonist of adenosine A2B receptor; The adenosine A2B receptor antagonist is PSB-603.

5. Use according to claim 4, characterized in that, The dosage concentration of PSB-603 is above 10 μM.

6. The application according to claim 4, characterized in that, The pruritus includes at least one of the following: uremic pruritus, chronic pruritus, and acute pruritus.

7. Use according to claim 4, characterized in that, The itching is histamine- or chloroquine-induced.

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