阅读说明：本技术 治疗g蛋白偶联受体介导的病症的组合物和方法 (Compositions and methods for treating G protein-coupled receptor mediated disorders ) 是由 X·董 C·瓦萨达 S·H·斯奈德 J·梅西翁 Y·程 N·利姆詹亚翁 于 2018-06-15 设计创作，主要内容包括：本发明涉及用于检测影响G蛋白偶联受体介导的病症的细胞和方法。本发明也涉及用于治疗药物副作用、自身免疫性疾病和瘙痒的方法。(The present invention relates to cells and methods for detecting conditions affecting G protein-coupled receptor-mediated disorders. The invention also relates to methods for treating drug side effects, autoimmune diseases and pruritus.)

1. A method of screening for an agent that modulates one or more MrgprX4 or MrgprX 3G protein-coupled receptor mediated conditions or diseases, comprising:

contacting one or more cells expressing MrgprX4 or MrgprX 3G protein-coupled receptor with a candidate agent; and

detecting a response of the one or more cells, thereby selecting the agent for evaluation to modulate the G protein-coupled receptor mediated disorder or disease.

2. The method of claim 1, wherein the response of the cell is detected as activation of the G protein-coupled receptor.

3. The method of claim 1 or 2, further comprising determining whether the candidate agent modulates the G protein-coupled receptor mediated disorder or disease.

4. The method according to any one of claims 1 to 4, wherein the MrgprX4 or MrgprX 3G protein-coupled receptor mediated disorder is selected from the group consisting of drug side effects, autoimmune diseases, multiple sclerosis, pain, itch, cholestatic itch, inflammation, malignant transformation, skin disorders, and wound healing.

5. The method of any one of claims 1 to 4, wherein the one or more cells are selected from immune cells, neural cells, and skin cells.

6. The method of any one of claims 1-5, wherein the one or more cells comprise dendritic cells.

7. The method of any one of claims 1 to 5, wherein the one or more cells comprise keratinocytes.

8. The method of any one of claims 1-7, wherein the one or more cells comprise primary sensory neurons in the dorsal root ganglion.

9. The method of any one of claims 1 to 8, wherein the response detected is an increase in intracellular calcium, or activation is also assessed by inositol phosphate testing.

10. The method of claim 9, wherein activation of MrgprX3 or MrgprX4 is detected by identifying an increase in intracellular calcium or by inositol phosphate detection.

11. A method of treating a G protein-coupled receptor mediated disorder in a subject, the method comprising:

administering to the subject an effective amount of an MrgprX3 antagonist and/or an MrgprA6 antagonist, thereby treating the G protein-coupled receptor mediated disorder.

12. The method of claim 11, wherein the G protein-coupled receptor mediated disorder is selected from pain, itch, cholestatic itch, inflammation, malignant transformation, skin disease, and/or wound healing.

13. The method of claim 11 or 12, wherein the antagonist comprises an antibody or fragment thereof, a binding protein, a polypeptide, or any combination thereof.

14. The method of any one of claims 11-13, wherein the antagonist comprises a small molecule or a nucleic acid molecule.

15. A method of treating a G protein-coupled receptor mediated disorder in a subject, the method comprising:

administering to the subject an effective amount of an MrgprX3 agonist and/or an MrgprA6 agonist, thereby treating the G protein-coupled receptor mediated disorder.

16. The method of claim 15, wherein the G protein-coupled receptor mediated disorder is selected from pruritus, cholestatic pruritus, inflammation, and/or skin disorders and/or wound healing.

17. The method of claim 15 or 16, wherein the agonist comprises an antibody or fragment thereof, a binding protein, a polypeptide, or any combination thereof.

18. The method of any one of claims 15-17, wherein the agonist comprises a small molecule or a nucleic acid molecule.

19. A method of treating a G protein-coupled receptor mediated disorder in a subject, the method comprising:

administering to the subject an effective amount of an MrgprX4 antagonist and/or an MrgprA1 antagonist, thereby treating the G protein-coupled receptor mediated disorder.

20. The method of claim 19, wherein the G protein-coupled receptor mediated disorder is selected from the group consisting of a drug side effect, an autoimmune disease, multiple sclerosis, pain, itch, and cholestatic itch.

21. The method of claim 19 or 20, wherein the antagonist comprises an antibody or fragment thereof, a binding protein, a polypeptide, or any combination thereof.

22. The method of any one of claims 19-21, wherein the antagonist comprises a small molecule or a nucleic acid molecule.

23. A method of treating a G protein-coupled receptor mediated disorder in a subject, the method comprising:

administering to the subject an effective amount of an MrgprX4 agonist and/or an MrgprA1 agonist, thereby treating the G protein-coupled receptor mediated disorder.

24. The method of claim 23, wherein the G protein-coupled receptor mediated disorder is selected from pain, itch, and cholestatic itch.

25. The method of claim 23 or 24, wherein the antagonist comprises an antibody or fragment thereof, a binding protein, a polypeptide, or any combination thereof.

26. The method of any one of claims 23-25, wherein the agonist comprises a small molecule or a nucleic acid molecule.

27. A method of reducing the severity of a drug side effect induced by an administered compound in a subject, the method comprising:

administering the compound to a subject;

administering an mrgprA1 antagonist and/or an mrgprX4 antagonist to the subject, thereby reducing the severity of drug side effects in the subject.

28. A method of determining whether a subject has an increased risk of developing a drug side effect for a compound, the method comprising:

obtaining a test sample from a subject having or at risk of developing a drug side effect on a compound;

determining the expression level of at least one G protein-coupled receptor gene within the test sample;

comparing the expression level of said G protein-coupled receptor gene in said test sample with the expression level of said G protein-coupled receptor gene in a reference sample; and

determining that administration of the compound to the patient will induce a drug side effect if the expression level of the G protein-coupled receptor gene indicated in the test sample differs from the expression level of the G protein-coupled receptor gene indicated in the reference sample.

29. The method of claim 28, wherein the G protein-coupled receptor gene is MrgprA1 or MrgprX 4.

30. The method of claim 29, wherein the MrgprA1 or MrgprX4 is a mutant.

31. A pharmaceutical composition for treating a G protein-coupled receptor mediated condition or disease, said composition comprising an effective amount of a G protein-coupled receptor antagonist.

32. The pharmaceutical composition of claim 31, wherein the G protein-coupled receptor antagonist is an MrgprX4 or MrgprX3 antagonist.

33. The pharmaceutical composition of claim 31 or 32, wherein the antagonist is selected from the group consisting of an antibody or fragment thereof, a binding protein, a polypeptide, a small molecule, a nucleic acid, or any combination thereof.

34. A pharmaceutical composition for treating a G protein-coupled receptor mediated condition or disease, said composition comprising an effective amount of a G protein-coupled receptor agonist.

35. The pharmaceutical composition of claim 34, wherein the G protein-coupled receptor antagonist is an MrgprX4 or MrgprX3 agonist.

36. The pharmaceutical composition of claim 34 or 35, wherein the agonist is selected from the group consisting of an antibody or fragment thereof, a binding protein, a polypeptide, a small molecule, a nucleic acid, or any combination thereof.

37. A kit, comprising: 1) the pharmaceutical composition of any one of claims 31-36, and 2) written instructions for use in treating the G protein-coupled receptor disorder or disease.

38. The kit of claim 37, wherein the written instructions for use are for treating a drug side effect, an autoimmune disease, multiple sclerosis, pain, itch, cholestatic itch, inflammation, malignant transformation, a skin disease, and/or wound healing.

39. An isolated cell comprising a recombinant nucleic acid that expresses mas-associated G-protein coupled receptor member X3(MrgprX3) or MrgprA 6.

40. The isolated cell of claim 39, wherein the recombinant nucleic acid expresses MrgprX 3.

41. The isolated cell of claim 39, wherein the recombinant nucleic acid expresses MMrgprA 6.

42. The isolated cell of claim 40, wherein the recombinant nucleic acid expressing MrgprX3 comprises one or more mutations.

43. The isolated cell of claim 42, wherein the one or more mutations result in a MrgprX3 protein that is unable to activate a signal transduction channel.

44. The isolated cell of claim 41, wherein the recombinant nucleic acid expressing MrgprA6 comprises one or more mutations.

45. The isolated cell of claim 44, wherein the one or more mutations result in MrgprA6 protein that is unable to activate a signal transduction channel.

46. The isolated cell of claim 41, wherein the isolated cell comprises a human embryonic kidney 293(HEK 293) cell, an innate immune cell, a stem cell, or a cell line.

47. An isolated cell comprising a recombinant nucleic acid that expresses mas-associated G-protein coupled receptor member X4(MrgprX4) or MrgprA 1.

48. The isolated cell of claim 47, wherein the recombinant nucleic acid expresses MrgprX 4.

49. The isolated cell of claim 47, wherein the recombinant nucleic acid expresses MrgprA 1.

50. The isolated cell of claim 48, wherein the recombinant nucleic acid expressing MrgprX4 comprises one or more mutations.

51. The isolated cell of claim 50, wherein the one or more mutations result in a MrgprX4 protein that is unable to activate a signal transduction channel.

52. The isolated cell of claim 49, wherein the recombinant nucleic acid expressing MrgprA1 comprises one or more mutations.

53. The isolated cell of claim 52, wherein the one or more mutations result in MrgprA1 protein that is unable to activate a signal transduction channel.

54. The isolated cell of claim 47, wherein the isolated cell comprises a human embryonic kidney 293(HEK 293) cell, an innate immune cell, a stem cell, or a cell line.

55. A method of identifying an antagonist of MrgprX3 or MrgprA6, the method comprising:

contacting the isolated cell of claim 39 with a compound that induces a skin or epithelial cell response,

contacting said isolated cell with a candidate antagonist, and

detecting activation of MrgprX3 or MrgprA6, wherein activation of MrgprX3 or MrgprA6 is reduced relative to activation of MrgprX3 or MrgprA6 in the absence of the compound, determining that the candidate compound is an antagonist.

56. A method of identifying an antagonist of MrgprX4 or MrgprA1, the method comprising:

contacting an isolated cell according to claim 47 with a compound that induces a drug side effect or an itch response,

contacting said isolated cell with a candidate antagonist, and

detecting activation of MrgprX4 or MrgprA1, wherein activation of MrgprX4 or MrgprA1 is reduced relative to activation of MrgprX4 or MrgprA1 in the absence of the compound, determining that the candidate compound is an antagonist.

57. A method of identifying a dual/multiple antagonist of MrgprX3, MrgprX4, or other Mrgpr member, the method comprising:

contacting an isolated cell of any of claims 39-5449 with a compound that induces a skin or epithelial cell response and a drug side effect or itch response, contacting the isolated cell with a candidate antagonist, and

detecting activation of MrgprX4 and MrgprX3, wherein activation of MrgprX4 and MrgprX3 is reduced relative to activation of MrgprX4 and MrgprX3 in the absence of the compound, determining that the candidate compound is a multiplex antagonist.

58. A method of identifying an agonist of MrgprX3 or MrgprA6, the method comprising:

contacting an isolated cell according to any one of claims 39 to 46 with a compound that induces a skin or epithelial cell response,

contacting said isolated cell with a candidate agonist, and

detecting activation of MrgprX3 or MrgprA6, wherein activation of MrgprX3 or MrgprA6 is increased relative to activation of MrgprX3 or MrgprA6 in the absence of the compound, determining that the candidate compound is an agonist.

59. A method of identifying an agonist of MrgprX4 or MrgprA1, the method comprising:

contacting an isolated cell according to any one of claims 47 to 54 with a compound that induces a drug side effect or an itch response,

contacting said isolated cell with a candidate agonist, and

detecting activation of MrgprX4 or MrgprA1, wherein activation of MrgprX4 or MrgprA1 is increased relative to activation of MrgprX4 or MrgprA1 in the absence of the compound, determining that the candidate compound is an agonist.

60. A recombinant nucleic acid that expresses mas-related G-protein coupled receptor member X3(MrgprX3) or MrgprA 6.

61. The recombinant nucleic acid of claim 60, wherein the recombinant nucleic acid expresses MrgprX 3.

62. The recombinant nucleic acid according to claim 60, wherein the recombinant nucleic acid expresses MrgprA 6.

63. The recombinant nucleic acid of claim 61, wherein the recombinant nucleic acid that expresses MrgprX3 comprises one or more mutations.

64. The recombinant nucleic acid of claim 63, wherein the one or more mutations produces MrgprX3 protein that is unable to activate a signal transduction channel.

65. The recombinant nucleic acid of claim 62, wherein the recombinant nucleic acid that expresses MrgprA6 comprises one or more mutations.

66. The recombinant nucleic acid of claim 65, wherein the one or more mutations produces MrgprA6 protein that is unable to activate a signal transduction channel.

67. A recombinant nucleic acid that expresses mas-related G-protein coupled receptor member X4(MrgprX4) or MrgprA 1.

68. The recombinant nucleic acid according to claim 67, wherein the recombinant nucleic acid expresses MrgprX 4.

69. The recombinant nucleic acid according to claim 67, wherein the recombinant nucleic acid expresses MrgprA 1.

70. The recombinant nucleic acid of claim 68, wherein the recombinant nucleic acid that expresses MrgprX4 comprises one or more mutations.

71. The recombinant nucleic acid of claim 70, wherein the one or more mutations produces MrgprX4 protein that is unable to activate a signal transduction channel.

72. The recombinant nucleic acid of claim 69, wherein the recombinant nucleic acid that expresses MrgprA1 comprises one or more mutations.

73. The recombinant nucleic acid of claim 72, wherein the one or more mutations produces MrgprA1 protein that is unable to activate a signal transduction channel.

74. A vector comprising a nucleic acid sequence encoding mas-related G protein-coupled receptor member X3(MrgprX3) or MrgprA 6.

75. The vector of claim 74, wherein the nucleic acid sequence encodes a MrgprX3 nucleic acid sequence comprising one or more mutations.

76. The vector of claim 74, wherein the nucleic acid sequence encodes a MrgprA6 nucleic acid sequence comprising one or more mutations.

77. A vector comprising a nucleic acid sequence encoding mas-related G protein-coupled receptor member X4(MrgprX4) or MrgprA 1.

78. The vector of claim 77, wherein the nucleic acid sequence encodes a MrgprX4 nucleic acid sequence comprising one or more mutations.

79. The vector of claim 77, wherein the nucleic acid sequence encodes a MrgprA1 nucleic acid sequence comprising one or more mutations.

Background

G protein-coupled receptor mediated diseases including chronic pruritus (e.g., pruritus), inflammation, autoimmunity, skin disorders, and drug side effects cause pain. The pathology of G protein-coupled receptor mediated diseases is largely unknown. There is a great need for the treatment of G protein-coupled receptor mediated diseases.

Disclosure of Invention

The present invention is based, in part, on the identification of novel G protein-coupled receptors, human MrgprX4 and murine MrgprA 1. MrgprX4 and MrgprA1 are expressed within specific types of innate immune cells, mediate Stevens-Johnson syndrome (SJS), and appear to be involved in autoimmune disease. MrgprX4 and MrgprA are activated by a variety of drugs that cause SJS including lamotrigine (lamotrigine) and allopurinol (allopurinol). In addition, MrgprX4 and MrgprA are also expressed within sensory neurons and are important for itching sensations and cholestatic pruritus. In some embodiments, MrgprX4 and MrgprA1 are receptors for bilirubin. As described herein, no bilirubin receptor has been identified prior to this finding. In some embodiments, human MrgprX4 is a drug target for SJS, autoimmune diseases such as multiple sclerosis, cholestatic pruritus, and other chronic pruritus. As described herein, prior to this finding, the role MrgprX4 plays in any biological process and disease was completely unknown. In some embodiments, assays based on cells expressing MrgprX 4(MrgprX4 cell line and cDNA, and MrgprA1 mutant murine lines) are used to screen and test drugs targeting these responses. As described herein, the MrgprX 4-expressing cell line is brand new and is used for high throughput screening for drug screening. In some embodiments, blocking MrgprX4 is treatment of SJS; autoimmune diseases such as multiple sclerosis; and novel approaches to cholestatic pruritus and other chronic pruritus.

In a preferred aspect, there is provided a method of screening for an agent that modulates one or more G protein-coupled receptor mediated conditions or diseases, the method comprising: (1) contacting one or more cells expressing a G protein-coupled receptor with a candidate agent; and (2) detecting a response of the one or more cells, thereby selecting the agent for evaluation to modulate a G protein-coupled receptor mediated disorder or disease. Suitably, the response of the cell is detected as activation of the G protein-coupled receptor. The method may further comprise determining whether the candidate agent modulates the G protein-coupled receptor mediated disorder or disease.

The present invention is also based in part on the discovery that human MrgprX3 and its murine congener mra 6 are expressed within primary sensory neurons in keratinocytes, epithelial cells, and Dorsal Root Ganglia (DRGs). Antimicrobial peptide defensins and antimicrobial peptides (cathelicidins) were also found to be agonists of MrgprX3 and MrgprA 6. Defensins and antimicrobial peptides may play a role in a variety of diseases and conditions including wound healing, chronic inflammation, malignant transformation, skin diseases such as psoriasis and dermatitis, respiratory diseases, digestive and gastrointestinal diseases, pain and itch. In some embodiments, MrgprX3 and MrgprA6 are targeted for the treatment of wound healing, chronic inflammation, malignant transformation, skin disorders such as psoriasis and dermatitis, respiratory disorders, digestive and gastrointestinal disorders, pain and itch. As described herein, the role that MrgprX3 plays in any biological process and disease is completely unknown. In some embodiments, agents targeting these responses are screened and tested using MrgprX 3-based cell-based assays and MrgprA6 mutant mice. As described herein, the MrgprX 3-expressing cell line is novel and useful for high throughput screening for drug screening.

Also provided herein are methods of screening for drugs targeting these receptors using assays based on cells expressing MrgprX3 and MrgprX 4. The invention also provides MrgprX3 and MrgprX4 expressing cell lines for high throughput screening of drug candidates. In some embodiments, blocking MrgprX4 treats drug side effects (e.g., SJS), cholestatic pruritus and other chronic pruritus, and autoimmune diseases (e.g., multiple sclerosis). In some embodiments, MrgprX3 is used to treat wound healing, chronic inflammation, malignant transformation, skin disorders such as psoriasis and dermatitis, respiratory and gastrointestinal disorders, pain and itch.

The invention is also based in part on an isolated cell comprising a recombinant nucleic acid that expresses mas-related G protein-coupled receptor member X3(MrgprX3) or MrgprX 4. For example, the recombinant nucleic acid expresses MrgprX 3. Alternatively, the recombinant nucleic acid expresses MrgprX 4. In other cases, a recombinant nucleic acid that expresses MrgprX3 comprises one or more mutations. For example, the one or more mutations result in MrgprX3 protein that is unable to activate a signal transduction channel. Alternatively, a recombinant nucleic acid expressing MrgprX4 comprises one or more mutations. For example, the one or more mutations result in MrgprX4 protein that is unable to activate a signal transduction channel. In some embodiments, the cell is selected from the group consisting of an immune cell, a neural cell, and a skin cell. In some embodiments, the immune cell is selected from an innate immune cell. In some embodiments, the cell is selected from a stem cell. In some embodiments, the cell is selected from a cell line. In some embodiments, the cell is a primary cell. In some embodiments, the cell is obtained from a mammal. In some embodiments, the neural cell consists of a primary sensory neuron in the dorsal root ganglion. In some embodiments, the immune cell consists of a dendritic cell. In some embodiments, mrpgx 4 and MrpgrA1 are expressed within dendritic cells and within primary sensory neurons in the dorsal root ganglia. In some embodiments, the skin cell is a keratinocyte.

The invention is also based in part on recombinant nucleic acids expressing mas-related G protein-coupled receptor member X3(MrgprX3) or MrgprX 4. For example, the recombinant nucleic acid is an expression vector and expresses MrgprX 3. Alternatively, the recombinant nucleic acid expresses MrgprX 4. In other cases, a recombinant nucleic acid that expresses MrgprX3 comprises one or more mutations. For example, the one or more mutations result in MrgprX3 protein that is unable to activate a signal transduction channel. Alternatively, a recombinant nucleic acid expressing MrgprX4 comprises one or more mutations. For example, the one or more mutations result in MrgprX4 protein that is unable to activate a signal transduction channel. In some embodiments, the vector comprises a nucleic acid sequence encoding mas-related G protein-coupled receptor member X3(MrgprX3) or MrgprA 6. In some embodiments, the vector comprises a nucleic acid sequence encoding MrgprX3 nucleic acid sequence comprising one or more mutations. In some embodiments, the vector comprises a nucleic acid sequence encoding MrgprA6 nucleic acid sequence comprising one or more mutations. In some embodiments, the vector comprises a nucleic acid sequence encoding mas-related G protein-coupled receptor member X4(MrgprX4) or MrgprA 1. In some embodiments, the vector comprises a nucleic acid sequence encoding MrgprX4 nucleic acid sequence comprising one or more mutations. In some embodiments, the vector comprises a nucleic acid sequence encoding MrgprA1 nucleic acid sequence comprising one or more mutations.

Provided herein is a method of screening for a drug that modulates a G protein-coupled receptor mediated disorder or disease, the method comprising: contacting a cell expressing a G protein-coupled receptor with a candidate agent; detecting G protein-coupled receptor-mediated activation; determining whether the candidate agent modulates a G protein-coupled receptor mediated disorder or disease. In some embodiments, the G protein-coupled receptor gene is selected from MrgprX4 and MrgprX 3. In some embodiments, the G protein-coupled receptor mediated disorder is selected from the group consisting of drug side effects, autoimmune diseases, multiple sclerosis, pain, itch, cholestatic itch, inflammation, malignant transformation, skin disorders, and wound healing. In some embodiments, the cell is selected from the group consisting of an immune cell, a neural cell, and a skin cell. In some embodiments, the immune cell is selected from an innate immune cell. In some embodiments, the cell is selected from a stem cell. In some embodiments, the cell is selected from a cell line. In some embodiments, the cell is a primary cell. In some embodiments, the cell is obtained from a mammal. In some embodiments, the neural cell consists of a primary sensory neuron in the dorsal root ganglion. In some embodiments, the immune cell consists of a dendritic cell. In some embodiments, mrpgx 4 and MrpgrA1 are expressed within dendritic cells and within primary sensory neurons in the dorsal root ganglia. In some embodiments, the skin cell is a keratinocyte. In some embodiments, activation of MrgprX3 or MrgprX4 is detected by identifying an increase in intracellular calcium.

Also provided herein are methods of treating a G protein-coupled receptor-mediated disorder in a subject, the method comprising administering an MrgprX3 antagonist and/or an MrgprA6 antagonist to the subject, thereby treating the G protein-coupled receptor-mediated disorder. In some embodiments, the G protein-coupled receptor mediated disorder is selected from the group consisting of pain, itch, cholestatic itch, inflammation, malignant transformation, skin disorders, and wound healing.

Also provided are methods of treating a G protein-coupled receptor mediated disorder in a subject, the method comprising administering a MrgprX3 antagonist and/or a MrgprA6 agonist to the subject, thereby treating the G protein-coupled receptor mediated disorder. In some embodiments, the G protein-coupled receptor mediated disorder is selected from the group consisting of pain, itch, cholestatic itch, inflammation, and skin disorders (e.g., psoriasis and allergic dermatitis).

In some embodiments of such methods, the antagonist or agonist comprises an antibody or fragment thereof, a binding protein, a polypeptide, or any combination thereof. In some embodiments, the antagonist or agonist comprises a small molecule. In some embodiments, an antagonist or agonist comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises double-stranded ribonucleic acid (dsRNA), small hairpin RNA, and short hairpin RNA (shrna), or antisense RNA, or any portion thereof. In some embodiments of such methods, the antagonist or agonist is administered prior to, concurrently with, or subsequent to the administration of the compound to the subject. In some embodiments, the antagonist or agonist is administered topically, orally, by inhalation, or by injection.

Also provided herein are methods of treating a G protein-coupled receptor-mediated disorder in a subject, the method comprising administering an MrgprX4 or MrgprA1 antagonist to the subject, thereby treating the G protein-coupled receptor-mediated disorder. In some embodiments of such methods, the G protein-coupled receptor-mediated disorder is selected from the group consisting of drug side effects such as stevens-johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN), autoimmune diseases, multiple sclerosis, pain, pruritus and cholestatic pruritus.

Also provided are methods of treating a G protein-coupled receptor mediated disorder in a subject, the method comprising administering an MrgprX4 or MrgprA1 agonist to the subject, thereby treating the G protein-coupled receptor mediated disorder. In some embodiments of such methods, the G protein-coupled receptor-mediated disorder is selected from the group consisting of a drug side effect, an autoimmune disease, pruritus, and cholestatic pruritus.

In some embodiments of such methods, the antagonist or agonist comprises an antibody or fragment thereof, a binding protein, a polypeptide, or any combination thereof. In some embodiments, the antagonist comprises a small molecule. In some embodiments, an antagonist or agonist comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises double-stranded ribonucleic acid (dsRNA), small hairpin RNA, and short hairpin RNA (shrna), or antisense RNA, or any portion thereof. In some embodiments, the antagonist or agonist is administered prior to, concurrently with, or subsequent to the administration of the compound to the subject. In some embodiments of such methods, the antagonist or agonist is administered topically, orally, by inhalation, or by injection.

Also provided herein is a method of reducing the severity of a drug side effect induced by an administered compound in a subject, the method comprising: administering the compound to a subject; administering an MrgprA1 or MrgprX4 antagonist or agonist to the subject, thereby reducing the severity of a drug side effect in the subject.

Also provided herein are methods of determining whether a subject has an increased risk of developing a drug side effect for a compound, the method comprising: obtaining a test sample from a subject having or at risk of developing a drug side effect on a compound; determining the expression level of at least one G protein-coupled receptor gene within the test sample; comparing the expression level of said G protein-coupled receptor gene in said test sample with the expression level of said G protein-coupled receptor gene in a reference sample; and determining that administration of the compound to the patient will induce a drug side effect if the expression level of the G protein-coupled receptor gene indicated in the test sample is different from the expression level of the G protein-coupled receptor gene indicated in the reference sample. In some embodiments, the G protein-coupled receptor gene is MrgprA1 or MrgprX 4. In some embodiments, MrgprA1 or MrgprX4 is a mutant.

Also provided herein are pharmaceutical compositions for treating a G protein-coupled receptor mediated disorder or disease, the compositions comprising an effective amount of a G protein-coupled receptor antagonist or agonist. In some embodiments, the G protein-coupled receptor antagonist is an MrgprX4 or MrgprX3 antagonist, or a dual/multiplex (dual/multivalent) antagonist of MrgprX3, MrgprX4, and other Mrgpr members. In certain other embodiments, the G protein-coupled receptor agonist is an MrgprX4 or MrgprX3 agonist. In some embodiments, the antagonist or agonist is selected from the group consisting of an antibody or fragment thereof, a binding protein, a polypeptide, a small molecule, a nucleic acid, or any combination thereof. In some embodiments, the antagonist or agonist is administered topically, orally, by inhalation, or by injection. In some embodiments, the G protein-coupled receptor-mediated disorder or disease is selected from the group consisting of drug side effects, autoimmune diseases, multiple sclerosis, pain, pruritus, cholestatic pruritus, inflammation, malignant transformation, skin disorders, and wound healing.

Also provided are kits comprising 1) the pharmaceutical compositions disclosed herein, and 2) written instructions for use in treating the G protein-coupled receptor disorder or disease. The pharmaceutical compositions may suitably comprise an effective amount of a G protein-coupled receptor antagonist such as MrgprX4 or MrgprX3 antagonist. In some embodiments, the pharmaceutical composition may suitably comprise an effective amount of a G protein-coupled receptor antagonist, such as MrgprX4 or MrgprX3 agonist. The written instructions for use can be, for example, a label or package insert that discloses methods of using the pharmaceutical compositions to treat, for example, a drug side effect, an autoimmune disease, multiple sclerosis, pain, itch, cholestatic itch, inflammation, malignant transformation, a skin disorder, and/or wound healing.

Also provided herein are compositions of isolated cells comprising a recombinant nucleic acid that expresses mas-related G protein-coupled receptor member X3(MrgprX3) or MrgprA 6. In some embodiments, the recombinant nucleic acid expresses MrgprX 3. In some embodiments, the recombinant nucleic acid expresses MrgprA 6. In some embodiments, a recombinant nucleic acid that expresses MrgprX3 comprises one or more mutations. In some embodiments, the one or more mutations result in MrgprX3 protein that is unable to activate a signal transduction channel. In some embodiments, a recombinant nucleic acid that expresses MrgprA6 comprises one or more mutations. In some embodiments, the one or more mutations result in MrgprA6 protein that is unable to activate a signal transduction channel. In some embodiments, the isolated cells include human embryonic kidney 293(HEK 293) cells.

Also provided herein are compositions of isolated cells comprising a recombinant nucleic acid that expresses mas-related G protein-coupled receptor member X4(MrgprX4) or MrgprA 1. In some embodiments, the recombinant nucleic acid expresses MrgprX 4. In some embodiments, the recombinant nucleic acid expresses MrgprA 1. In some embodiments, a recombinant nucleic acid that expresses MrgprX4 comprises one or more mutations. In some embodiments, the one or more mutations result in MrgprX4 protein that is unable to activate a signal transduction channel. In some embodiments, a recombinant nucleic acid that expresses MrgprA1 comprises one or more mutations. In some embodiments, the one or more mutations result in MrgprA1 protein that is unable to activate a signal transduction channel. In some embodiments, the isolated cells include human embryonic kidney 293(HEK 293) cells.

Also provided herein are methods of identifying an antagonist of MrgprX3 or MrgprA6, comprising: contacting an isolated cell (e.g., an isolated cell comprising a recombinant nucleic acid that expresses mas-related G protein-coupled receptor member X3(MrgprX3) or MrgprA6) with a compound that induces a pseudo-allergic type response, contacting the isolated cell with a candidate antagonist, detecting activation of MrgprX3 or MrgprA6, wherein activation of MrgprX3 or MrgprA6 is reduced relative to activation of MrgprX3 or MrgprA6 in the absence of the compound, determining that the candidate compound is an antagonist.

Also provided are methods of identifying an agonist of MrgprX3 or MrgprA6, the method comprising: contacting an isolated cell (e.g., an isolated cell comprising a recombinant nucleic acid that expresses mas-related G protein-coupled receptor member X3(MrgprX3) or MrgprA6) with a compound that induces a drug side effect, contacting the isolated cell with a candidate agonist, detecting activation of MrgprX3 or MrgprA6, wherein activation of MrgprX3 or MrgprA6 is increased relative to activation of MrgprX3 or MrgprA6 in the absence of the compound (i.e., a control), determining that the candidate compound is an agonist. Preferably, a candidate agonist increases activation of MrgprX3 or MrgprA6 by at least 1,2, 3, 4, or 5 percentage points relative to a test in the absence of the candidate agonist (control), more preferably, a candidate agonist increases activation of MrgprX3 or MrgprA6 by at least 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 percentage points relative to the same test in the absence of the candidate agonist (control). Preferred test assays for evaluating candidate agonists include the assay of example 9, after which activation can be assessed by calcium imaging or inositol phosphate detection.

Also provided herein are methods of identifying an antagonist of MrgprX4 or MrgprA1, comprising: contacting an isolated cell (e.g., an isolated cell comprising a recombinant nucleic acid that expresses mas-related G protein-coupled receptor member X4(MrgprX4) or MrgprA1) with a compound that induces a drug side effect, contacting the isolated cell with a candidate antagonist, detecting activation of MrgprX4 or MrgprA1, wherein activation of MrgprX4 or MrgprA1 is reduced relative to activation of MrgprX4 or MrgprA1 in the absence of the compound (i.e., a control), and determining that the candidate compound is an antagonist. Preferably, the candidate antagonist reduces activation of MrgprX4 or MrgprA1 by at least 1,2, 3, 4, or 5 percentage points relative to a test in the absence of the candidate antagonist (control), more preferably, the candidate antagonist reduces activation of MrgprX4 or MrgprA1 by at least 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 percentage points relative to the same test in the absence of the candidate antagonist (control). Preferred test assays for evaluating candidate antagonists include the assay of example 9, after which activation can be assessed by calcium imaging detection. Activation can also be assessed by inositol phosphate detection.

Also provided are methods of identifying an agonist of MrgprX4 or MrgprA1, the method comprising: contacting an isolated cell (e.g., an isolated cell comprising a recombinant nucleic acid that expresses mas-related G protein-coupled receptor member X4(MrgprX4) or MrgprA1) with a compound that induces a drug side effect, contacting the isolated cell with a candidate agonist, detecting activation of MrgprX4 or MrgprA1, wherein activation of MrgprX4 or MrgprA1 is increased relative to activation of MrgprX4 or MrgprA1 in the absence of the compound (i.e., a control), determining that the candidate compound is an agonist. Preferably, a candidate agonist increases activation of MrgprX4 or MrgprA1 by at least 1,2, 3, 4, or 5 percentage points relative to a test in the absence of the candidate agonist (control), more preferably, a candidate agonist increases activation of MrgprX4 or MrgprA1 by at least 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 percentage points relative to the same test in the absence of the candidate agonist (control). Preferred test assays for evaluating candidate agonists include the assay of example 9, after which activation can be assessed by calcium imaging or inositol phosphate detection.

Provided herein are methods of reducing the severity of a G protein-coupled receptor-mediated disorder induced by administration of a compound in a subject, the method comprising: administering the compound to a subject; administering to the subject a MrgprX4 antagonist, a MrgprX3 antagonist, or a combination thereof, thereby reducing the severity of a G protein-coupled receptor-mediated disorder in the subject.

Also provided is a method of reducing the severity of a G protein-coupled receptor-mediated condition induced by administration of a compound in a subject, the method comprising: administering the compound to a subject; administering a MrgprX4 agonist, a MrgprX3 agonist, or a combination thereof to the subject, thereby reducing the severity of a G protein-coupled receptor-mediated disorder in the subject.

Also provided is a method of reducing the severity of a drug side effect induced by an administered compound in a subject, the method comprising: administering the compound to a subject; administering an MrgprX4 antagonist to the subject, thereby reducing the severity of drug side effects in the subject.

Also provided are methods of reducing the severity of wound healing, chronic inflammation, malignant transformation, skin diseases such as psoriasis and dermatitis, respiratory and gastrointestinal disorders, pain or itch in a subject; administering an MrgprX3 antagonist to the subject, thereby reducing the severity of wound healing, chronic inflammation, malignant transformation, skin diseases such as psoriasis and dermatitis, respiratory and gastrointestinal disorders, pain and/or itch in the subject.

Also provided are methods of reducing the severity of wound healing, chronic inflammation, malignant transformation, skin diseases such as psoriasis and dermatitis, respiratory and gastrointestinal disorders, pain or itch in a subject; administering an MrgprX3 agonist to the subject, thereby reducing the severity of wound healing, chronic inflammation, malignant transformation, skin diseases such as psoriasis and dermatitis, respiratory and gastrointestinal disorders, pain and/or itch in the subject.

For example, the methods described herein prevent or reduce the severity of a G protein-coupled receptor mediated disorder by at least 1%, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

The subject is preferably a mammal in need of such treatment or prophylaxis, such as a subject who has been diagnosed with a pseudoallergy type reaction or predisposition thereto. The mammal is any mammal, for example, humans, primates, mice, rats, dogs, cats, horses, and domestic or food animals such as cattle, sheep, pigs, chickens, and goats. In a preferred embodiment, the mammal is a human.

The inhibitor or antagonist or agonist may include, but is not limited to, nucleic acids, peptides, antibodies, or small molecules that bind to their natural ligand at a specific target or targets and modulate biological activity.

In some cases, the antagonist or agonist comprises a small molecule. Small molecules are compounds with a mass of less than 2000 daltons. The small molecule preferably has a molecular mass of less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound has a molecular mass of less than 500 daltons, less than 400 daltons, less than 300 daltons, less than 200 daltons or less than 100 daltons.

Small molecules are organic or inorganic. Exemplary small organic molecules include, but are not limited to, aliphatic hydrocarbons, alcohols, aldehydes, ketones, organic acids, esters, monosaccharides, disaccharides, aromatic hydrocarbons, amino acids, and lipids. Exemplary inorganic small molecules include trace minerals, ions, free radicals, and metabolites. Alternatively, small molecules can be synthetically engineered to consist of fragments, or small portions, or longer amino acid chains to fill the binding pocket of the enzyme. Typical small molecules are less than one kilodalton.

In some cases, the antagonist or agonist comprises a nucleic acid molecule. For example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) inhibits expression of MrgprX3 or MrgprX4, thereby inhibiting the activity of MrgprX3 or MrgprX 4. In some cases, the nucleic acid comprises small interfering RNA (sirna), RNA interference (RNAi), messenger RNA (mrna), small hairpin RNA or short hairpin RNA (shrna), double-stranded ribonucleic acid (dsRNA), antisense RNA or microRNA, or any portion thereof. However, one of skill in the art will readily identify other nucleic acids that inhibit/antagonize or activate/agonize MrgprX3 or MrgprX 4.

As previously described, the antagonist or agonist may be an antibody, for example a monoclonal or polyclonal MrgprX3 or MrgprX4 antibody. For example, MrgprX3 or MrgprX4 antibodies that can be used as antagonists include monoclonal polyclonal antibodies, such as murine, rabbit, primate (e.g., monkey) or humanized antibodies (e.g., the commercially available rabbit polyclonal MrgprX4 antibody from Novus Biologicals, accession number NLS 2429; rabbit polyclonal MrgprX4 antibody b97784 from Abcam; rat polyclonal MrgrpX4 antibody ab188740 from Abcam; and the anti-MrgprX 3 polyclonal antibody PA5-3395 from Thermo Fischer). Fragments of such monoclonal antibodies may also be suitable antagonists or agonists, including fragments of the labeled commercially available antibodies. Suitable and preferred antibody fragments for use as MrgprX3 or MrgprX4 antagonists or agonists are readily identifiable by the assays disclosed herein. Suitable fragments may have a sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% identical to the sequence of the corresponding antibody, e.g., an annotated commercially available antibody. Such fragments may be intact agents that act as antagonists or agonists of MrgprX3 or MrgprX4, or may be covalently linked to another sequence or other molecule, for example to form a fusion molecule containing the antibody fragment sequences, or containing sequences having suitable sequence identity to the corresponding antibodies, as noted for commercially available antibodies.

As also discussed herein, suitable and preferred MrgprX4 and MrgprX3 antagonists and agonists, including small molecules, polypeptides, antibodies and antibody fragments, and nucleic acids, can be readily identified, including by the assays disclosed herein.

Administration of the antagonist or agonist is prior to, concurrent with, or subsequent to administration of the compound to the subject.

A variety of routes of administration may be used. For example, the antagonist or agonist is administered topically, orally, by inhalation, or by injection.

An effective amount of an antagonist or agonist is 0.001mg/kg to 250mg/kg body weight, e.g., 0.001mg/kg, 0.05mg/kg, 0.01mg/kg, 0.05mg/kg, 1mg/kg, 5mg/kg, 10mg/kg, 25mg/kg, 50mg/kg, 75mg/kg, 100mg/kg, 125mg/kg, 150mg/kg, 175mg/kg, 200mg/kg, 225mg/kg, or 250mg/kg body weight. Basically, the attending physician or veterinarian will determine the appropriate amount and dosage regimen.

In some cases, the dosage regimen of the antagonist or agonist is at least once daily, at least once weekly, or at least once monthly. The antagonist or agonist may be suitably administered continuously for one day, one week, one month, two months, three months, six months, nine months or one year. In some cases, the antagonist is administered daily, e.g., every 24 hours. Alternatively, the antagonist is administered continuously or several times daily, e.g., once every 1 hour, once every 2 hours, once every 3 hours, once every 4 hours, once every 5 hours, once every 6 hours, once every 7 hours, once every 8 hours, once every 9 hours, once every 10 hours, once every 11 hours, or once every 12 hours.

The method of determining whether a compound induces a drug side effect is performed by: contacting the isolated cells described herein with a candidate compound and detecting activation of MrgprX4, wherein activation of MrgprX4 identifies the candidate compound as inducing a drug side effect.

For example, activation of MrgprX3 or MrgprA6 is identified by an increase in intracellular calcium levels relative to the absence of the compound. In some cases, the calcium level in the cell is increased by at least 1%, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. The intracellular calcium concentration is determined using the methods described herein or those available to those skilled in the art.

For example, activation of MrgprX4 or MrgprA1 is identified by an increase in intracellular calcium levels relative to the absence of the compound. In some cases, the calcium level in the cell is increased by at least 1%, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. The intracellular calcium concentration is determined using the methods described herein or those available to those skilled in the art.

Candidate MrgprX4 antagonists are screened for evidence of countering or inhibiting, reducing, or suppressing the biological activity of MrgprX4 polypeptides. Candidate MrgprX3 antagonists are screened for evidence of countering or inhibiting, reducing, or suppressing the biological activity of MrgprX3 polypeptides.

Also provided are methods of identifying an antagonist of MrgprX4 or MrgprX3, the method comprising: contacting an isolated cell described herein with a compound that induces a pseudo-allergic type response, contacting an isolated cell described herein with a candidate antagonist, and detecting activation of MrgprX3 or MrgprX4, wherein activation of MrgprX3 or MrgprX4 is reduced relative to activation of MrgprX3 or MrgprX4 in the absence of the candidate antagonist, determining that the candidate compound is an antagonist. Preferably, the candidate antagonist reduces activation of MrgprX3 or MrgprX4 by at least 1,2, 3, 4, or 5 percentage points relative to a test in the absence of the candidate antagonist (control), more preferably, the candidate antagonist reduces activation of MrgprX3 or MrgprX4 by at least 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 percentage points relative to the same test in the absence of the candidate antagonist (control). Preferred test assays for evaluating candidate antagonists include the assay of example 9, after which activation can be assessed by calcium imaging or inositol phosphate detection.

Also provided are methods of identifying an agonist of MrgprX4 or MrgprX3, comprising: contacting an isolated cell described herein (e.g., an isolated cell comprising a recombinant nucleic acid that expresses mas-related G protein-coupled receptor member X3(MrgprX3) or MrgprX4) with a compound that induces a pseudo-allergic type response, contacting an isolated cell described herein with a candidate agonist, detecting activation of MrgprX3 or MrgprX4, wherein activation of MrgprX3 or MrgprX4 is increased relative to activation of MrgprX3 or MrgprX4 in the absence of the candidate agonist, determining that the candidate compound is an agonist. Preferably, the selected candidate agonist reduces activation of MrgprX3 or MrgprX4 by at least 1,2, 3, 4, or 5 percentage points relative to the test in the absence of the candidate agonist (control), more preferably, the candidate agonist reduces activation of MrgprX3 or MrgprX4 by at least 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 percentage points relative to the same test in the absence of the candidate agonist (control). Preferred test assays for evaluating candidate agonists include the assay of example 9, after which activation can be assessed by calcium imaging or inositol phosphate detection.

Also provided herein are methods of treating an autoimmune disease (e.g., multiple sclerosis) in a subject, the method comprising: identifying a subject that is suffering from or at risk of developing an autoimmune disease, and administering to the subject an effective amount of a composition comprising an MrgprX4 antagonist, thereby treating or preventing the autoimmune disease (e.g., multiple sclerosis) in the subject.

Exemplary autoimmune diseases are selected from the group consisting of: celiac disease, type 1 diabetes, Graves disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus (SLE or lupus).

Also provided herein are methods of treating wound healing in a subject, the method comprising: identifying a subject undergoing wound healing or at risk of undergoing wound healing, and administering to the subject an effective amount of a composition comprising an MrgprX3 antagonist, thereby treating or assisting wound healing in the subject.

Also provided herein are methods of treating a skin disorder in a subject, the method comprising: identifying a subject suffering from or at risk of developing a skin disorder, and administering to the subject an effective amount of a composition comprising an MrgprX3 antagonist, thereby treating or preventing the skin disorder in the subject.

Also provided is a method of treating a skin disorder in a subject, the method comprising: identifying a subject suffering from or at risk of developing a skin disorder, and administering to the subject an effective amount of a composition comprising an agonist of MrgprX3, thereby treating or preventing the skin disorder in the subject.

Exemplary skin diseases treated by such methods include psoriasis, dermatitis, skin ulcers, and cancer (e.g., melanoma).

Also provided herein are methods of treating inflammation (e.g., chronic inflammation) in a subject, the method comprising: identifying a subject that is suffering from or at risk of developing inflammation, and administering to the subject an effective amount of a composition comprising an MrgprX3 antagonist, thereby treating or preventing inflammation (e.g., chronic inflammation) in the subject.

Exemplary inflammations are selected from the group consisting of: chronic inflammation, appendicitis, bursitis, colitis, cystitis, dermatitis, phlebitis, reflex sympathetic dystrophy/complex regional pain syndrome (rsd/crps), rhinitis, tendonitis, tonsillitis, acne vulgaris, reactive respiratory diseases such as asthma and respiratory infections, autoimmune diseases, autoinflammatory diseases, celiac disease, chronic prostatitis, diverticulitis, glomerulonephritis, hidradenitis suppurativa, allergies, bowel diseases (including intestinal epithelial diseases such as inflammatory bowel diseases such as irritable bowel syndrome and colitis), interstitial cystitis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection and vasculitis.

Also provided herein are methods of treating a malignant transformation (e.g., cancer) in a subject, the method comprising: identifying a subject that is suffering from or at risk of developing a malignant transformation, and administering to the subject an effective amount of a composition comprising an MrgprX3 antagonist, thereby treating or preventing the malignant transformation (e.g., cancer) in the subject.

Exemplary cancers are selected from the group consisting of: carcinomas, sarcomas, tumors, solid tumors, blood cancers, leukemias, lymphatic cancers, skin cancers, melanomas, breast cancers, ovarian cancers, uterine cancers, prostate cancers, testicular cancers, colorectal cancers, stomach cancers, intestinal cancers, bladder cancers, lung cancers, non-small cell lung cancers, pancreatic cancers, renal cell cancers, kidney cancers, liver cancers, hepatocellular cancers, brain cancers, head and neck cancers, retinal cancers, gliomas, lipomas, laryngeal cancers, thyroid cancers, neuroblastoma, endometrial cancers, myeloma, and esophageal cancers.

The compositions described herein are administered via: oral administration, intravenous administration, external administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, nasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intraarterial administration, intraarticular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppository administration, inhalation administration, or subcutaneous administration.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide the skilled artisan with a general definition of a number of terms used in the present invention: cambridge scientific Dictionary (The Cambridge Dictionary of Science and Technology (Walkered, 1988)); rieger et al, written in The Glossary of Genetics,5th Ed, R.Rieger et al, (eds.), Springer Verlag (1991); and The "Halper-Coriolis biological Dictionary" by Hale and Marham (Hale & Marham, The Harper Collins Dictionary of Biology (1991)), as used herein, The following terms have The meanings ascribed below unless expressly excluded.

Antibodies and fragments thereof described herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single chain antibodies, Fab fragments, Fab 'fragments, F (ab')2 fragments, Fv and scFv. Fragments of an antibody possess the immunological activity of its counterpart antibody. In some embodiments, a fragment of an antibody contains 1500 or fewer, 1250of less,1000 or fewer, 900 or fewer, 800 or fewer, 700 or fewer, 600 or fewer, 500 or fewer, 400 or fewer, 300 or fewer, 200 or fewer amino acids. For example, a protein or peptide inhibitor contains 1500 or less, 1250 or less,1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less,100 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less,10 or less amino acids. For example, the nucleic acid inhibitors of the invention contain 400 or less, 300 or less, 200 or less, 150 or less,100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 35 or less, 30 or less, 28 or less, 26 or less, 24 or less, 22 or less, 20 or less, 18 or less, 16 or less, 14 or less, 12 or less,10 or less nucleotides.

General methods in molecular and cellular biochemistry can be found in standard texts, such as molecular cloning: a Laboratory Manual (fourth edition) (Molecular Cloning: A Laboratory Manual,4th Ed. (Sambrook et al, Cold spring Harbor Laboratory Press 2012)); short protocols in Molecular Biology,5th Ed, (Ausubel et al eds., John Wiley & Sons 2002); protein Methods (Bollag et al, John Wiley & Sons 1996)); non-viral Vectors for Gene Therapy (non viral Vectors for Gene Therapy (Wagner et al eds., Academic Press 1999)); viral Vectors (Kaplift & Loewy eds., academic Press 1995)); a Manual of immunological Methods (Immunology Methods Manual (I.Lefkovits ed., Academic Press 1997)); and "cell and tissue culture: biotechnology Laboratory Procedures (Cell and tissue culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998)). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

As used herein, the term "antibody" (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. Herein, the term "immunoglobulin" (Ig) is used interchangeably with "antibody".

An "isolated antibody" is an antibody that has been isolated from and/or recovered from a component of its natural environment. Contaminant components of the natural environment of an antibody are materials that would interfere with the diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is purified: (1) to more than 95% by weight of antibody, as measured by the Lowry method, and most preferably more than 99% by weight; (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid by using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE staining using Coomassie blue or preferably silver under reducing or non-reducing conditions. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Typically, however, the isolated antibody will be prepared by at least one purification step.

The antibody four-chain basic unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 such heterotetrameric basic units together with an additional peptide called J chain and thus contain 10 antigen binding sites, while secreted IgA antibodies can aggregate to form multivalent assemblies comprising 2 to 5 four chain basic units and J chains. In the case of IgG, the four chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by a monovalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H-chain and L-chain also has regularly spaced interchain disulfide bridges. Each H chain has a variable domain (V) at the N-terminus_H) For the α chain and the gamma chain, the V_HAfter the domainFollowed by three constant domains (C)_H) For the mu isoforms and isoforms, the V_HField followed by four C_HA domain. Each L chain has a variable domain (V) at the N-terminus_H) Then is located at the V_HConstant domain at the other end of the domain (C)_L)。V_LAnd V_HAlignment, and C_LTo the first constant domain of the heavy chain (C)_H1) And (6) aligning. It is believed that particular amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. Will V_HAnd V_LPaired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology (eighth edition), 8th edition, DanielP.Stits, Abba I.Terr and Tristram G.Parslow (eds.), Appleton&Lange, Norwalk, conn.,1994) page 71, chapter 6.

Based on its constant domain (C)_L) The L chain from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ). According to the constant domain of its heavy chain (C)_H) There are five types of immunoglobulins, IgA, IgD, IgE, IgG and IgM, with heavy chains designated alpha (α), delta (), epsilon (), gamma (γ) and mu (μ), respectively_HWith relatively minor differences in sequence and function, the γ and α classes are further divided into subclasses, e.g., humans express subclasses of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2.

The term "variable" refers to the fact that: the sequences of certain fragments of the V domains of different antibodies vary widely. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acid span of the variable domain. Instead, the V region consists of a number of relatively invariant stretches called Framework Regions (FRs) separated by short, extremely variable regions called "hypervariable regions", which are 15 to 30 amino acids in length, with each hypervariable region being 9 to 12 amino acids in length. The variable domains of native heavy and light chains each comprise four FRs, predominantly in the β -sheet configuration, connected by three hypervariable regions which form loop junctions and, in some cases, form part of the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FR and with the hypervariable regions from the other chain, contributing to the formation of the antigen-binding site of the antibody (see, "protein Sequences of Immunological Interest (fifth edition)", (Kabat et al, Sequences of Proteins of Immunological Interest,5th ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991))). Constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit multiple effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions typically comprise CDRs from a "complementarity determining region" or "CDR" (e.g., V when counted according to the Kabat counting system_LAbout around residues 24 to 34(L1), 50 to 56(L2) and 89 to 97(L3), V_HAbout at residues 31 to 35(H1), residues 50 to 65(H2) and residues 95 to 102 (H3); amino acid residues of immunologically significant protein Sequences (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991))); and/or those from the "hypervariable loop" (e.g., V when counted according to the Chothia counting system_LResidues 24 to 34(L1), residues 50 to 56(L2) and residues 89 to 97(L3) in (1), and V_HResidues 26 to 32(H1), residues 52 to 56(H2) and residues 95 to 101(H3) in (a); residues of Chothia and Lesk, J.mol.biol.196:901-917 (1987)); and/or those from "hypervariable loops"/CDRs (e.g., V when counted according to the IMGT counting system_LResidues 27 to 38(L1), residues 56 to 65(L2) and residues 105 to 120(L3) in (1), and V_HResidues 27 to 38(H1), residues 56 to 65(H2) and residues 105 to 120(H3) in (a); lefranc, M.P.et al.Nucl.acids Res.27:209-212 (1999); ruiz, M.e al.Nucl.AcidsRes.28:219-221 (2000)). Optionally, the antibody has a symmetric insertion at one or more of the following points: when based on AHo; hV when counted by onneger, A.and Plunkthun, A.J.mol.biol.309:657-_L28, 36(L1), 63, 74 to 75(L2) and 123(L3), and V_H28, 36(H1), 63, 74 to 75(H2) and 123 (H3).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Furthermore, unlike polyclonal antibody preparations that include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. The advantages of the monoclonal antibodies are, in addition to their specificity, that they can be synthesized by other antibodies without impurities. The modifier "monoclonal" is to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention can be prepared by the hybrid cell method first described in Kohler et al, Nature,256:495(1975), or can be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No.4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al, Nature,352: 624-.

Monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences derived from another species or belonging to another antibody class or subclass, fragments of such antibodies, so long as they exhibit the desired biological activity (see, U.S. Pat. No.4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA,81: 6851-. Also provided are variable domain antigen binding sequences derived from human antibodies. Accordingly, chimeric antibodies of most interest herein include antibodies having one or more human antigen binding sequences (e.g., CDRs) and containing one or more sequences derived from a non-human antibody, such as FR or C region sequences. Furthermore, chimeric antibodies of most interest herein are those comprising a human variable domain antigen binding sequence of one antibody class or subclass and another sequence derived from another antibody class or subclass, such as an FR or C region sequence. Chimeric antibodies of interest herein also include those that contain variable domain antigen binding sequences related to those described herein or derived from a different species, such as a non-human primate (e.g., old world monkey, ape, etc.). Chimeric antibodies also include primatized (primatized) and humanized antibodies.

Furthermore, the chimeric antibody may comprise residues not found in the recipient antibody or in the donor antibody. These modifications were made to further refine antibody potency. For further details see Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-; and Presta, curr, Op, Structure, biol.2:593-596 (1992).

A "humanized antibody" is generally considered to be a human antibody having one or more amino acid residues from a non-human source introduced into the antibody. These non-human amino acid residues are generally referred to as "import" residues, which are typically taken from an "import" variable domain. Traditionally, humanization has been performed by replacing the corresponding sequences of human antibodies with input hypervariable region sequences, following the method of Winter and co-workers (Jones et al, Nature,321:522-525 (1986); Reichmann et al, Nature,332:323-327 (1988); Verhoeyen et al, Science,239:1534-1536 (1988)). Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which substantially less than an entire human variable domain has been replaced with the corresponding sequence from a non-human species.

A "human antibody" is an antibody that contains only the sequences present in an antibody naturally produced by a human. However, as used herein, a human antibody may comprise residues or modifications not found in human antibodies that occur to humans, including those modifications and variant sequences described herein. Typically, these modifications are made to enhance antibody potency.

An "intact" antibody is one that comprises an antigen binding site and a CL and at least heavy chain constant domains CH1, CH 2 and CH 3. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')2, and Fv fragments; a bispecific antibody; linear antibodies (see, U.S. Pat. No. 5,641,870; Zapata et al, Protein Eng.8(10):1057-1062[1995 ]); a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

The phrase "functional fragment or analog" of an antibody is a compound that has the same qualitative biological activity as a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is a compound that can bind to an IgE immunoglobulin in a manner that prevents or substantially reduces the molecule from having binding to the high affinity receptor FcThe possibility of RI capability.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and the remaining "Fc" fragment, a digestion that reflects the ability to crystallize readily. The Fab fragment consists of the entire L chain together with the variable domain of the H chain (VH), and the first constant domain of one heavy chain (CH 1). With respect to the antigenic sites, each Fab fragment is monovalent, i.e., each Fab fragment has one antigen binding site. Pepsin treatment of an antibody results in a single large F (ab')2 fragment which roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen binding activity and which still can crosslink antigen. Fab 'fragments differ from Fab fragments in that Fab' fragments also have a small number of residues at the carboxy terminus of the CH1 domain including one or more cysteines from the hinge region of the antibody. Herein, Fab '-SH refers to Fab' whose cysteine residues of the constant domains carry a free thiol group. F (ab ')2 antibody fragments were originally produced as pairs of Fab ' fragments with a hinge cysteine between the two Fab ' fragments. Other chemical couplings of antibody fragments are also known.

The "Fc" fragment comprises the carboxy-terminal portions of two H chains held together by disulfide bonds. The effector functions of an antibody are determined by sequences within the Fc region, which is also the portion recognized by Fc receptors (fcrs) found on certain types of cells.

"Fv" is the smallest antibody fragment containing the complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region and one light chain variable region in close, non-covalent association. The folding of these two domains creates 6 hypervariable loops (3 loops from each of the H and L chains), which contribute amino acid residues for antigen binding and provide antigen binding specificity to the antibody. However, even a single variable domain (or a half Fv comprising only 3 CDRs specific for an antigen) has the ability to recognize and bind an antibody, even if the affinity of the binding is lower than the entire binding site.

"Single chain Fv", also abbreviated as "SFv" or "scFv", comprises a VH antibody domain and a VL antibody domain linked as a single chain of a polypeptide. Preferably, the sFv polypeptide further comprises a polypeptide chain linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in Roche burg (Rosenburg) and Moore, eds Monoclonal antibody Pathology 113 (The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994)); borebaeck 1995, infra.

The term "diabodies" refers to small antibody fragments prepared by: sFv fragments with short linkers (about 5 to 10 residues) between the VH and VL domains are constructed (see preceding paragraphs) to allow inter-chain but not intra-chain pairing of multiple V domains, resulting in bivalent fragments, i.e., fragments with two antigen binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are disclosed more fully in EP 404,097, WO 93/11161 and Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-.

As used herein, an "internalizing" antibody is an antibody that is taken up by (i.e., enters) a mammalian cell upon binding to an antigen on the cell (e.g., the cell expresses a polypeptide or receptor). Of course, internalizing antibodies will include antibody fragments, human or chimeric antibodies, and antibody conjugates. For certain therapeutic applications, internalization in vivo is contemplated. The number of antibody molecules internalized will be sufficient or appropriate to kill or inhibit the growth of cells, particularly infected cells. Depending on the potency of the antibody or antibody conjugate, in some cases, uptake of a single antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds. For example, a toxin has a high potential for killing such that internalization of a molecule from coupling the toxin to the antibody is sufficient to kill the infected cell.

As used herein, the affinity constant K of an antibody is preferred if it reacts at a detectable level with the antigen_aGreater than or equal to about 10⁴M^-1Or greater than or equal to about 10⁵M^-1Or greater than or equal to about 10⁶M^-1Or greater than or equal to about 10⁷M^-1Or greater than or equal to about 10⁸M^-1The antibody is said to be "immunospecific," specific for, or "specifically binds" to an antigen. The affinity of an antibody for its cognate antigen is also generally expressed as the dissociation constant KD, in certain embodiments if HuM2e antibody is present at less than or equal to 10^-4M, less than or equal to 10^-5M, less than or equal to 10^-6M, less than or equal to 10^-7M, or less than or equal to 10^-8K of M_DWhen bound to M2e, HuM2e specifically binds to M2 e. The affinity of the antibody can be readily determined using conventional techniques such as those disclosed in Scatchard et al (Ann.N.Y.Acad.Sci.USA51:660 (1949)).

The binding properties of antibodies to antigens, cells or tissues thereof can generally be determined using immunodetection methods including, for example, immunofluorescence-based assays such as Immunohistochemistry (IHC) and/or Fluorescence Activated Cell Sorting (FACS).

An antibody having a "biological characteristic" of a given antibody is one that has an acquired biological characteristic that distinguishes the antibody from other antibodies. For example, in certain embodiments, an antibody having the biological characteristics of a given antibody will bind to the same epitope as the given antibody, and/or have common effector functions as the given antibody.

The term "antagonist" antibody is used in the broadest sense and includes an epitope, polypeptide or cell whose biological activity specifically binds is partially or completely blocked, inhibited or neutralized. The method of identifying an antagonist antibody can comprise: contacting a polypeptide or cell specifically bound by the candidate antagonist antibody with the candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the polypeptide or cell.

Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region) and vary with antibody isotype. Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); autophagy; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

The term "antigen binding site" or "binding site" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") region of the heavy ("H") chain and the light ("L") chain. Three highly differentiated stretches within the V regions of the heavy and light chains, called "hypervariable regions", are inserted between the more conserved flanking stretches called "framework regions" or "FRs". Thus, the term "FR" refers to an amino acid sequence naturally found between and adjacent to hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged with respect to each other within a three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface to which the antigen is bound, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs".

As used herein, the term "epitope" includes any moiety capable of

A protein determinant that specifically binds to an immunoglobulin, scFv or T cell receptor. Epitopic determinants consist of chemically active surface components of the molecule, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an antibody can be directed against the N-terminus or C-terminus of a polypeptide, a linear or nonlinear peptide sequence of a protein, and an epitope comprising amino acids of a first antigen and amino acids of a second antigen.

As used herein, the term "immune cell" generally includes white blood cells (leukocytes) derived from Hematopoietic Stem Cells (HSCs) produced in the bone marrow. "immune cells" include, for example, lymphocytes (T cells, B cells, Natural Killer (NK) cells) and myeloid cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). In some embodiments, the immune cell comprises a chimeric antigen receptor. As used herein, the term "chimeric antigen receptor" or "CAR" refers to an antigen binding domain fused to an intracellular signaling domain capable of activating or stimulating an immune cell, and in certain embodiments, the CAR further comprises a transmembrane domain.

As used herein, the terms "immunological binding" and "immunological binding characteristics" refer to the type of non-covalent interaction between an immunoglobulin molecule and an antigen specific for the immunoglobulin. The strength or affinity of the immunological binding effect may be the dissociation constant (K) of the effect_d) Expression of, among others, the smaller K_dIndicating greater affinity. The immunological binding properties of the selected polypeptide can be quantified using methods well known in the art. One such method entails measuring the rate of antigen-binding site/antigen complex formation and dissociation, which rates depend on the concentration of the complex members, the affinity of the action, and geometric parameters that affect the rates equally in both directions. Thus, the "association rate constant (K) can be determined by calculating the concentration and the actual rate of association and dissociation_on) "and" decomposition rate constant (K)_off)”。(Nature 361:186-87(1993))。K_off/K_onThe ratio of which is such that it is eliminatedAll affinity-independent parameters were possible and equal to the dissociation constant K_d. Davies et al (1990) Annual Rev Biochem 59: 439-. An antibody of the invention is said to specifically bind to an antigen or epitope described herein (e.g., CTLA, PD1, PDL1, or other immunosuppressive protein and/or tumor antigen) when the equilibrium binding constant (Kd), as measured by an assay such as a radioligand binding assay or similar assays known to those skilled in the art, is ≦ 1 μ M, preferably ≦ 100nM, more preferably ≦ 10nM, more preferably ≦ 1nM, and most preferably ≦ 100pM to about 1 pM.

The invention also encompasses polypeptide fragments and nucleic acid fragments so long as they exhibit the desired biological activity of the full-length polypeptide and nucleic acid, respectively (i.e., against MrgprX3 or MrgprX 4). Nucleic acid fragments of almost any length are used. For example, various implementations of the invention include exemplary polynucleotide segments having a total length of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length (including all intermediate lengths). Likewise, polypeptide fragments of almost any length are employed. For example, exemplary polypeptide segments having a total length of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 5,000, about 1,000, about 500, about 200, about 100, about 50 amino acids in length (including all intermediate lengths) are included in various implementations of the invention.

The polynucleotide, polypeptide, or other agent is purified and/or isolated. In particular, herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein, when produced by recombinant techniques, is substantially free of other culture materials or culture media; when chemically synthesized, are substantially free of chemical precursors or other chemicals. The purified compound contains at least 60% by weight (dry weight) of the compound of interest. Preferably, the formulation contains at least 75%, more preferably at least 90%, most preferably 99% by weight of the compound of interest. For example, a purified compound contains, by weight, at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound. Purity is measured by any suitable standard method, for example, column chromatography, thin layer chromatography or High Performance Liquid Chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) does not contain genes or sequences that flank it in its natural state. The purified or isolated polypeptide does not contain the amino acids or sequences that flank it in its native state. Purification also defines a degree of sterility that is safe for administration to a human subject, e.g., the absence of infectious or toxic agents.

Likewise, "substantially pure" means a nucleotide or polypeptide that has been separated from its naturally associated components. Typically, nucleotides and polypeptides are substantially pure when at least 60%, 70%, 80%, 90%, 95%, or even 99% by weight of the nucleotides and polypeptides are free of their naturally associated proteins and naturally occurring organic molecules.

By "isolated nucleic acid" is meant a nucleic acid that does not contain genes that flank the nucleic acid in the naturally occurring genome of the organism from which the nucleic acid is derived. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA but is not flanked by two nucleic acid sequences which flank the molecule in the genome of the organism from which it is naturally derived; (b) a nucleic acid that is incorporated into the genomic DNA of a vector or prokaryotic or eukaryotic cell in a manner such that the resulting molecule is distinct from the naturally occurring vector or genomic DNA; (c) an isolated molecule, such as synthetic complementary DNA (cDNA), a genomic fragment, a fragment produced by Polymerase Chain Reaction (PCR), or a restriction enzyme fragment; and (d) a recombinant nucleotide sequence which is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the invention further include molecules produced synthetically, as well as any nucleic acid that has been chemically altered and/or has a modified backbone. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide. Isolated nucleic acid molecules also include messenger ribonucleic acid (mRNA) molecules.

The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it is replicated and expressed. The term also refers to certain biological vectors, e.g., viral vectors and bacteriophages, that can be used for the same purpose, both infectious agents being capable of introducing heterologous nucleic acid sequences.

An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specific nucleic acid components that permit transcription of a particular polynucleotide sequence in a host cell. The expression vector may be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression vector includes a polynucleotide to be transcribed operably linked to a promoter. Summarizing in this context, "operably linked" means that two or more genetic elements, e.g., polynucleotide coding sequences and promoters, are placed in relative positions that allow the elements to perform an appropriate biological function, e.g., the promoter directs transcription of the coding sequence. As used herein, the term "promoter" refers to an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes essential nucleic acid sequences near the start site of transcription, e.g., in the case of a polymerase II type promoter, the TATA component. Promoters also optionally include distal enhancer or repressor components, which can be positioned as much as several thousand base pairs from the transcription start site. Other components that may be present in an expression vector include components that enhance transcription (e.g., enhancers) and components that terminate transcription (e.g., terminators), as well as components that confer some binding affinity or antigenicity to the recombinant protein produced from the expression vector.

"candidate compound" means a naturally occurring or artificially derived chemical. Candidate compounds can include, for example, peptides, polypeptides, synthetic organic molecules, natural organic molecules, nucleic acid molecules, peptide nucleic acid molecules, and components and derivatives thereof.

The term "pharmaceutical composition" means any composition that contains at least one therapeutically or biologically active agent and is suitable for administration to a patient. Any of these formulations can be prepared by methods well known and accepted in the art. See, for example, "Remington: pharmaceutical Science and Practice (twentieth edition) (Remington: The Science and Practice of pharmacy,20th edition, (ed.a.r.gennaro), Mack Publishing co., Easton, Pa., 2000).

By "G protein-coupled receptor (GPCR)" is meant a protein receptor that senses molecules outside the cell and activates signal transduction pathways within the cell and ultimately activates cellular responses. GPCRs are called seven transmembrane receptors because they cross the cell membrane seven times.

By "agonist" is meant a chemical substance that binds to the receptor and activates the extraction to produce a biological response. An "antagonist" blocks b the activity of an agonist as opposed to the agonist causing activity, and an inverse agonist causes activity that is in the opposite direction of the agonist activity. As used herein, the terms "antagonist" and "inhibitor" are used interchangeably to refer to any molecule that counters or inhibits, reduces or suppresses the biological activity of its target molecule. In some embodiments, an agonist is a "superagonist" when it induces or increases the biological activity of its target molecule (e.g., MrgprX4 or MrgprX 3). In some embodiments, an antagonist is a "super antagonist" when the antagonist counterbalances or inhibits, reduces or suppresses the biological activity of its target molecule (e.g., MrgprX4 or MrgprX 3). Suitable MrgprX3 antagonists, MrgprX4 antagonists, MrgprX3 agonists, and/or MrgprX4 agonists include soluble receptors, peptide inhibitors, small molecule inhibitors, ligandbodies, and antibodies.

"wild-type" or "WT" means the typical form of phenotype that a species presents in nature. Alternatively, the wild-type is conceptualized as the standard product, i.e., the "normal" allele at the locus, as opposed to the "mutant" allele of the non-standard producer.

As used herein, the term "administering" refers to any mode of transferring, delivering, introducing, or transporting MrgprX3 or MrgprX4 antagonist or MrgprX3 or MrgprX4 agonist, for example, to a subject in need of treatment for a disease or disorder. Such modes include, but are not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, nasal, and subcutaneous administration.

By "MrgprX 3 or MrgprX4 antagonist" is meant any small molecule, chemical compound, antibody, nucleic acid molecule or polypeptide, or fragment thereof, that is capable of blocking, preventing, reducing or altering the ability of MrgprX3 or MrgprX4 to activate a signal transduction pathway.

By "MrgprX 3 or MrgprX4 agonist" is meant any small molecule, chemical compound, antibody, nucleic acid molecule or polypeptide, or fragment thereof, that is capable of increasing, activating or altering the ability of MrgprX3 or MrgprX4 to activate a signal transduction channel. An MrgprX3 agonist or an MrgprX4 agonist can be identified by a protocol disclosed herein, e.g., example 9 below.

By "alteration" is meant a change (increase or decrease) in the activity of a polypeptide, e.g., MrgprX3 or MrgprX4, as detected by standard methods in the art such as those described herein. As used herein, alteration includes a change in the expression level or activity of a gene or polypeptide of 10% or more, preferably a change of 25%, more preferably a change of 40%, and most preferably a change of 50% or more in the activity of the polypeptide.

As used herein, "altering" also includes a 2-fold or more change in the expression level of a gene or polypeptide, e.g., 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold or more.

By "alleviating" is meant reducing, suppressing, attenuating, reducing, delaying or stabilizing the development or progression of a disease, e.g., a pseudo-allergic type of response.

"amplification" means increasing the number of copies of a molecule. In one example, the nucleic acid is amplified using the Polymerase Chain Reaction (PCR).

"binding" means having a physicochemical affinity for a molecule. Binding is measured by any method of the invention, e.g., binding of a drug/compound to a receptor expressed on a cell.

In this disclosure, "comprise," "comprise," and "have" and the like can have the meaning ascribed to them as set forth in U.S. patent law, and can be intended to mean "include" and the like; "consisting essentially of, and the like, have the meaning dictated by united states patent law, and the term is open ended, permitting the presence of more than the recited one, provided that the basic or novel features recited therein are not altered by the presence of more than the recited one, but do not include prior art embodiments.

By "detecting" is meant identifying, directly or indirectly, the presence, absence or amount of activation of MrgprX3 or MrgprX4 of the signal transduction channel to be detected.

By "effective amount" is meant the amount of compound required to alleviate the symptoms of the disease relative to an untreated patient. The effective amount of active compound for the therapeutic treatment of diseases to be used in the practice of the present invention will vary depending on the mode of administration and the age, weight and general health of the subject. Basically, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as the "effective" amount.

As used herein, the term "treating" (or "treatment") refers to administering an agent or formulation to a clinically symptomatic individual having a negative symptom, disorder or disease to achieve a reduction in the severity and/or frequency of the symptom, to eliminate the symptom and/or its underlying cause, and/or to facilitate amelioration or remediation of the injury.

Ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from any group consisting of 1,2, 3, 4,5, 6, 7, 8,9, 10, 11,12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimals between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to "sub-ranges," nested sub-ranges extending from either end of the range are particularly contemplated. For example, nested subranges of the exemplary ranges 1 to 50 can include 1 to 10,1 to 20, 1 to 30, and 1 to 40 in one direction, and 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in another direction.

"recombination" means a nucleic acid molecule formed by the laboratory process of genetic recombination (e.g., molecular cloning) bringing together genetic material from multiple sources to create sequences that cannot be found within a biological organism.

A "heterologous promoter" is a promoter that is different from the promoter to which a gene or nucleic acid sequence is operably linked in nature. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects the transcription and/or translation of the nucleic acid corresponding to the second sequence. As used herein, a "heterologous polynucleotide" or "heterologous gene" is a polynucleotide or gene that originates from a source distinct from the particular host cell, or, if from the same source, is modified from its original form.

By "decrease" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

"reference" means standard or control conditions.

The terms "a" and "an," as used herein, are to be construed as referring to the singular or the plural, unless expressly stated or apparent from the context. As used herein, the term "or" is understood to be inclusive, unless explicitly indicated or evident from the context.

Unless explicitly stated or otherwise evident from the context, the term "about" as used herein is understood to be within the normal tolerance of the art, e.g., within 2 standard deviations of the mean. "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. All numbers provided herein are modified by the term "about" unless explicitly excluded from the context.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments of the invention and from the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI documents identified by accession numbers cited herein are incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Drawings

Fig. 1A to E are a series of illustrations, graphs, photographs and micrographs demonstrating that Lamotrigine (Lamotrigine) binds directly to murine Mrgpra1 and its human homolog MRGPRX 4. FIG. 1A is a schematic of the loci showing the mouse and human Mrgpr genes. Murine Mrgpra3, Mrgprc11, and their human homologs, MRGPRX1, are specifically expressed within sensory neurons in the Dorsal Root Ganglia (DRGs) and function as itch receptors in response to itch substances including Chloroquine (CQ) and BAM8-22 (BAM). Murine Mrgprb2 and its human homolog MRGPRX2 are receptors for basic secretagogues (e.g., compound 48/80 and PAMP), are specifically expressed in mast cells, and mediate drug-induced allergic reactions. The MRGPRX4 homolog Mrgpra1 was described in this study. . Saal1, Ptpn5 and Zdhhc13 are unrelated genes within the genome. FIG. 1E is a graph showing that LTG induces internalization of Mrgpra 1-GFP. Dye-labeled LTG can create bright Red fluorescence (Texas Red). Starving serum with Medium, stained Medium, LTG, stained LTG ((S))>4hs) Mrgpra1-GFP cells were treated at 37 ℃ for 15 min. Staining showed that LTG to be stained was internalized into Mrgpra1-GFP cells. FIG. 1C is a graph showing elution profiles on MRGPRX 2/Cell Membrane Chromatography (CMC) columns and MRGPRX4/CMC columns. LTG could not be retained on the MRGPRX2/CMC column. The retention time of lamotrigine on the MRGPRX4/CMC column was 16.7 minutes. FIGS. 1C and 1D are graphs showing MRGPRX4/CMC penetration curves for LTG (FIG. 1C) and passage 1/[ LR ]]s to 1/[ L ]]m (fig. 1D) plot of the regression curve plotted against bar mean ± SEM (n ═ 5) points in fig. 1D, lamotrigine concentrations of 2.0 × 10 and 10 respectively⁸、4.0×10-⁸、8.0×10-⁸、1.6×10-⁷And 3.2×10-⁷mol/l.kd lamotrigine ═ (4.17 ± 0.24) × 10^-7mol/L. Each result was repeated more than three times.

Fig. 2A-2F are a series of exemplary illustrations, photographs, micrographs and graphs demonstrating that LTG can induce a SJS-like phenotype in 129S1/SvlmJ WT mice, but not in Mrgpra1KO mice. Figure 2A is an illustration demonstrating how mice with Mrgpra1 open reading frame replaced with GFP were generated. Fig. 2B is a photograph demonstrating that after 7 to 10 days of oral administration of 50mg/kg body weight LTG, WT mice developed mucosal secretions in their eyes and blebs on the paw, similar to the symptoms seen in patients who were bitter to SJS, whereas Mrgpra1KO mice did not. FIGS. 2C and 2E are photographs and graphs demonstrating that WT and HET Mrgpra1 were observed 7 days after LTG treatment^+/GFPMice developed conjunctival secretions, which was not seen in WT mice treated with saline or KO mice treated with LTG. H&E staining showed local tissue defects and some inflammatory cell infiltration near the epithelial-conjunctival junction in WT and HET mice, whereas saline treated mice and KO mice did not. The conjunctiva of WT and HET mice also had increased staining for the apoptotic cell marker TUNEL (LTG WT: 40.45. + -. 2.58%; LTG HET: 40.39. + -. 1.45%), whereas saline-treated mice and KO mice did not exhibit this increased cell death (saline WT: 7.08. + -. 1.99%; LTG KO: 13.56. + -. 1.74%), (, p)<0.01). Fig. 2D and 2F are photographs and graphs demonstrating that at day 9 of LTG treatment, symptoms of WT and HET mice further developed, which showed significant paw edema and bleb (fig. 2D), whereas saline treated mice and KO mice did not. H&E staining showed extensive infiltration of erythrocytes and inflammatory cells in the skin of WT and HET mice, which was not observed in saline treated mice and KO mice. The tissue within the black dashed rectangle is enlarged in fig. 6A to 6F. TUNEL assay detected a number of dead epithelial cells (green) in WT mice and HET mice (LTG WT: 48.74 + -5.94%; LTG HET: 45.99 + -1.84%), (. star, p)<0.01), whereas saline-treated mice and KO mice did not (saline WT: 10.56 +/-1.45%; LTG KO: 6.10 ± 1.63%). The scale bar is 100 μm in FIG. 6F and 50 μm in FIGS. 6C and 6D. FIG. 2F is a diagram showing the followingELISA results show quantification of granzyme B and TNF- α expression in paw skin 9 days after LTG treatment<0.01 (each genotype n ═ 5).

Fig. 3A-3F are a series of charts, photographs, micrographs, FACS plots, and immunoblots demonstrating that Mrgpra1 is expressed within a subset of Dendritic Cells (DCs) that play a key role in the development of the SJS phenotype. FIG. 3A is an immunoblot showing that RT-PCR showed that Mrgpra1 was expressed only in the spleen and lymph nodes of WT mice, but not in KO mice. FIG. 3B is a cytotaxogram demonstrating flow cytometry demonstrating GFP⁺The cell is the dendritic cell marker CD11c⁺And MHCII⁺. FIGS. 3C and 3D are immunoblots and micrographs showing the separation of CD11C from the spleen of WT and KO mice⁺And MHCII^{Height of}Cell, CD11c^-And MHCII^-Cell, CD11c⁺And MHCII^intCell, CD11c^-And MHCII⁺Cells, RT-PCR detection (fig. 3C) and calcium imaging (fig. 3D) were performed. As shown in FIG. 3C, RT-PCR revealed WT murine CD11C alone⁺And MHCII^{Height of}The cells express Mrgpra 1. As shown in FIG. 3D, calcium imaging showed that four different cells detached from the spleen of WT mice responded at 10 seconds (before LTG addition), 60 seconds (after LTG addition), and 180 seconds (after LTG washing). The yellow arrows indicate that LTG induces CD11C⁺And MHCII^{Height of}Two of the cells [ Ca ] at 60 seconds²⁺]_iAnd (4) increasing. The scale bar is 25 μm. The right panels are the respective imaging tracings. Each line is a response from a unique cell. CD11C only⁺And MHCII^{Height of}Cells had a good response to LTG (0.05 mg/L). Figure 3E is a graph showing calcium imaging assays (n-3 per genotype, counts per experiment)>100 cells, a, p<0.01) in the sample. FIG. 3F is a photograph showing that 2,500 ten thousand MHCII isolated from WT mice or KO mice⁺And CD11c⁺Dendritic Cells (DCs) were injected via tail vein into Mrgpra1KO mice (n ═ 6 per group). 7 days after LTG (50mg/kg body weight) intake, conjunctival secretions formed in mice receiving WT DCs. Mice receiving A1KO DC did not have any phenotype.

FIGS. 4A to 4F are a series of graphs, photographsSlides, micrographs, and immunoblots, indicating that human dendritic cells express MRGPRX4 and can be activated by LTG. FIG. 4A is an immunoblot showing RT-PCR results for human dendritic cells (hDC) showing that MRGPRX4 is expressed in hDC. FIG. 4B is a graph showing corresponding images from immunostaining of hDC treated with medium, stained medium, LTG (3 μ M) and stained LTG (3 μ M) for 15 minutes at 37 ℃. LTG (labeled with a dye, red) is internalized only into cells expressing MRGPRX4 (green). The scale bar is 100 μm. FIG. 4C is a graph showing that [ Ca ] is displayed from human dendritic cells (hDC) exposed to 0.1mg/L LTG (duration indicated by black line)²⁺]_iVarying tracer examples, measured by ratio Fura-2 imaging. Each trace is a response from a single cell. Fig. 4D is a micrograph showing the percentage of responsive cells from hdcs treated with LTG after receiving control siRNA (non-functional siRNA) and MRGPRX4 siRNA. Human DCs were transfected with siRNA against MRGPRX4 or control siRNA. After 48 hours, cells were treated with LTG and calcium imaged. hDC activation was significantly reduced in MRGPRX4 siRNA-treated cells compared to the control group. Each set of data was expressed as mean ± s.e.m. Significance of statistical comparisons was determined using a two-tailed unpaired student's t-test (n-4 per genotype; counts per experiment)>100 cells. A, p<0.01). Figure 4E shows that analysis of MRGPRX4 gene from SJS patients and tolerant populations shows that MRGPRX 4G/G mutations are more likely to occur in SJS patients. The percentage of G/G mutations in the tolerant population was 7.14% compared to 50% in SJS patients.

Fig. 5 is a series of photographs and graphs demonstrating the establishment of a SJS animal model. 129S1/SvImJ WT mice were gavaged with SJS-inducing drugs such as lamotrigine (50mg/kg body weight), oxcarbazepine (200mg/kg body weight), and allopurinol (100mg/kg body weight) for 20 days per day. After 7 days, in three groups, mice developed conjunctival secretions in their eyes. The highest incidence of mucosal secretions was induced by LTG (93.33%).

Fig. 6A to 6F are a series of charts and micrographs illustrating damage induced by LTG. FIG. 6A is a graph showing the change in body weight, which shows the weight loss (WT: -3.89. + -. 0.47 g; HET: -4.48. + -. 0.89g) in WT mice and HET mice after 10 days of treatment with LTG, while the weight gain (WT: 0.84. + -. 0.3 g; KO: 1.07. + -. 0.69g) in saline and KO groups. Fig. 6B is a graph showing the survival ratio showing the survival rate of mice after 14 days of LTG treatment. After 14 days, only 30% of the mice survived in the LTG-treated WT and HET groups, while none of the mice died in the saline and KO groups. Fig. 6C is a micrograph showing H & E staining showing subcutaneous tissue within the WT and KO rectangles shown in fig. 2D under a microscope using 40 x lenses. Erythrocytes and inflammatory cells may be found only in the WT dermis. The scale bar is 50 μm. Fig. 6D is a micrograph showing immunofluorescence double staining demonstrating CD 8-positive cells and CD 3-positive cells in the mouse eyelids after 9 days of treatment with LTG. The scale bar is 50 μm. FIG. 6E is a graph showing the number of CD8 positive cells and CD3 positive cells counted in the eyelids (native WT: 3.67 + -1.20, LTG WT: 25 + -8.34, LTG HET: 25 + -2, LTG KO: 5.67 + -0.88;, p < 0.01; n ═ 5 per genotype). Fig. 6F is a micrograph showing IHC staining of mouse paw intradermal granzyme B after 9 days of treatment with LTG and saline. Blue shading shows the localization of granzyme B. The scale bar is 100 μm.

FIG. 7 is a series of photographs showing Balb/c WT mice and Rag1(-/-) mice that received oral LTG (50mg/kg body weight) for 20 days. The photographs show the phenotype of the mice at days 7, 9 and 20. On day 7, Balb/c WT mice underwent the formation of mucosal secretions in their eyes. On day 9, WT mice developed blebs within their paw. On day 20, the ears of WT mice appeared peeled and thickened roughly.

Fig. 8A-8C are a series of illustrations, FACS plots, and photographs showing the role CD4 and CD 8T cells play during LTG treatment. Fig. 8A is an illustrative illustration showing the following treatment regimen: 129S 1WT mice received 3 intraperitoneal injections of rat monoclonal antibodies against murine CD4 and CD8 on days-2, 0 and 3, respectively. The mice were then treated with LTG for 14 days after receiving the second antibody injection. Fig. 8B is a graph of flow cytometry analysis demonstrating the absence of CD4 or CD 8T cells in vivo. On days 7 and 14, peripheral blood and spleen samples from the mice were collected for flow cytometry analysis. Fig. 8C is a photograph of mice undergoing LTG treatment. On day 9, mice receiving saline injection (left panel, labeled "WT") developed conjunctival secretions in their eyes and blebs in their paw, but these phenotypes were not found in the CD8 and CD 4T cell depleted groups.

Figures 9A to 9C are a series of flow cytometric images and micrographs showing that Mrgpra1 cells are CD11C and MHC-II positive cells. Fig. 9A is a flow cytometric image showing replacement of the Mrgpra1 open reading frame of Mrgpra1KO mice with GFP. Flow cytometry was used to characterize GFP + cells from Mrgpra1GFP/GFP mice; splenocytes and lymph nodes were stained with different antibodies such as CD4, CD8a, CD11b, CD45, CD317, CD370, F4/80, I-a/I-E, Ly6C, Ly6G, XCR1, CD3, and CD11 c. Subsequently, cell acquisition was performed on an LSR-II flow cytometer (BD Biosciences). The results showed that GFP + cells highly expressed CD11c and MHC-II. FIG. 9B is a micrograph showing immunofluorescence staining showing GFP cells are CD11c in A1KO murine spleen⁺Cells other than CD3⁺A cell. The scale bar is 50 μm. FIG. 9C is a diagram of flow cytometric analysis of lymph node cells, showing that Mrgpra1GFP/GFP cells are MHC-II⁺And CD11c⁺。

FIG. 10 is a series of FACS graphs showing the classification of different types of splenocytes. Based on CD11c and MHC-II expression, the immune cell populations were classified using FACS Diva software. The sorted cells were then immediately used for RNA isolation or calcium imaging.

FIG. 11 is a graph showing the body weight change of mice injected with WT mice and A1KO murine dendritic cells. LTG was used daily to treat a1KO mice, after which the mice received one injection of WT mice and a1KO murine Dendritic Cells (DCs). Starting on day 7, mice receiving WTDC experienced weight loss, while mice receiving a1KO DC experienced weight gain.

Fig. 12 is a series of micrographs showing the expression of MRGPRX4 in human dendritic cells. Immunofluorescence double staining of DAPI (blue) and MRGPRX4 (green) showed that MRGPRX4 was expressed within human dendritic cells. HEK293 cells and MRGPRX2-HEK cells were used as negative controls, while MRGPRX4-HEK cells were used as positive controls. The scale bar is 200 μm.

Fig. 13A-13F are a series of graphs showing that Mrgpr cluster KO mice have less scratching in a model of cholestatic pruritus. FIG. 13A is a graph showing the results of rats orally administered vehicle (olive oil) or 25mg/kg ANIT daily. On the fifth day, spontaneous itching was evaluated. The mice in the test chamber were videotaped for 30 minutes and the number of scratching events was counted. The mice are all littermates of 8 to 12 week old males. Blind studies on genotypes were performed and classified throughout the course of treatment. For each treatment and each genotype, n was 10, but position 6 in the cluster KO group treated with vehicle. Fig. 13B is a graph showing the results when serum was collected by cardiac puncture after evaluation of pruritus. Total bilirubin was measured by the animal pathology laboratory at JHU. Fig. 13C is a graph showing the results of liver weight after treatment. Fig. 13D is a graph showing the serum bile acid value results. Control n was 4, WT treated n was 10, and KO treated n was 7. Fig. 13E is a graph showing the results of serum autosomal chemokine activity (autotaxin). Control n was 4, WT treated n was 12 and KO treated n was 8 fig. 13F is a graph showing the results for serum methionine enkephalin values. Control n was 4, WT treated n was 19, and KO treated n was 10.

FIG. 14A is a series of graphs showing that bilirubin causes itching in WT mice but not in cluster KO mice. The black bars are WT mice. The red column is a cluster KO mouse. Yellow bars are MrgprA1 single gene knockouts. No itching source caused by injection is generated in the empty space. The number of scratching actions occurred was assessed 30 minutes after injection. The mice used in this study were littermates of 8 to 12 weeks of age. During the classification process, blind studies on treatment were performed. Mean values with SEM are shown. Figure 16A is a graph showing the results of subcutaneous injections of bilirubin in a vehicle (pH 6.6 to 7.0) containing 5% DMSO at the back of the neck of mice. FIG. 14B is a graph showing the results of mice injected on the back of the neck with 50 μ l of a 1mg/kg solution of morphine in saline. FIG. 14C is a graph showing the results of injecting 50 μ l of a 1mg/kg solution of DAMGO in saline into the back of the neck of a mouse. Fig. 14D is a graph showing the results of buccal injection of mice with the noted dose of bilirubin. Figure 14E is a graph showing the results of buccal injection of 1.3mM DCA in mice. Fig. 14F is a graph showing the results of buccal injection of 4mM LPA in mice.

Fig. 15A-15D are a series of graphs demonstrating that bilirubin activates a population of dorsal root ganglion neurons in an Mrgpr-dependent manner. FIG. 15A is a fluorescent intensity trace in which DRG neurons from WT mice were loaded with Fluo-4 AM calcium dye. Vehicle (0.5% DMSO) was added to neurons. After 30 seconds, a wash of 1 minute was applied. 50 μ M bilirubin was added. 50mM KCl was used as a positive control. Each trace represents a single neuron. Fig. 15B is a fluorescence intensity trace with bilirubin applied at 10 seconds. At 20 seconds, the bath solution was replaced with a 3mM EGTA bath solution. During this time bilirubin is again applied. Figure 15C is a graph showing vehicle or 50 μ M bilirubin applied to WT and cluster KO DRG neurons. During imaging, a neuron score is "activated" if its peak fluorescence reaches at least a 50% increase from baseline or 50% of the KCl peak. Furthermore, the neuron needs to have a signal above baseline of at least 20 seconds. Motion artifacts were excluded from the analysis. The total number of neurons from at least three mice was summed to calculate the percentage. Fig. 15D is a histogram showing neuron diameters scored "activated".

Fig. 16A-16C are a series of graphs demonstrating that bilirubin-mediated itch is non-histaminergic. Fig. 16A is a graph of histamine release when bilirubin is applied to mast cells as compared to the control group. Fig. 16B is a fluorescence intensity trace where application of bilirubin to peritoneal mast cells in the form of a bath failed to cause calcium influx. FIG. 16C is a bar graph of the results when 1mM bilirubin was injected into WT animals. The black bars are animals injected with vehicle. The red bar is 30mg/kg cetirizine (cetirizine), which is an H1R blocker.

Fig. 17A to 17F are a series of graphs showing activation of MrgprA1 and MrgprX4 by bilirubin in a G α q-dependent manner. Figure 17A is a graph demonstrating results when HEK cells that did not express MrgprA1 were loaded with Fura-2 calcium dye. 50 μ M bilirubin was added. After 30 seconds, a1 minute wash was applied and the cells were returned to baseline. Followed by another 50 μ M bilirubin addition. Each trace represents a single HEK cell. Fig. 17B is a graph showing the results when U73122(G α q retarder) was used. Fig. 17C is a graph showing results when U73343 (a closely related analog of U73122, without function against G α q) was used. Fig. 17D is a graph showing the results of the same experiment as fig. 17A but with HEK cells stably expressing MrgprX 4. 50 μ M bilirubin was applied in one application. Each trace represents a single cell. Fig. 17E is a graph showing the results of the same experiment as fig. 17B but using X4. Fig. 19F is a graph showing the results of the same experiment as fig. 17C but using X4.

Figures 18A to 18C are a series of graphs demonstrating that MrgprA1 and MrgprX4 have EC50 for bilirubin at pathophysiological concentrations. EC50 was calculated using HEK cells stably expressing MrgprA1 or MrgprX 4. Cells were loaded with FLIPR calcium imaging dye and changes in fluorescence were read using a Flexstation3 machine (Molecular Devices). Triplicate experiments were performed per well, and all values from each plate were normalized to the highest response well. Addition of vehicle alone did not cause a change in fluorescence of either cell line. Untransfected cells exhibited some degree of fluorescence change, but no change in dose response curve (not shown). The x-axis shows the modified logarithmic scale. Fig. 18A is a graph showing EC50 for MrgprA1 for approximately 49 μ M bilirubin. Fig. 20B is a graph showing EC50 for MrgprX4 for approximately 3 μ M bilirubin. Fig. 18C is a graph demonstrating that untransfected HEK cells do not show an EC50 response.

Fig. 19A to 19C are a series of graphs demonstrating MrgprA1 activation that causes pruritus. Injections of 1mM coproletin, 1mM hemoglobin or 1mM FMRF caused itching in WT animals, but not in cluster KO animals. All three compounds are agonists of MrgprA1 but not other Mrgpr agonists.

Fig. 20A to 20C are a series of graphs showing that MRGPRX3 is a novel keratinocyte receptor with BD. FIG. 20A is a series of corresponding Ca's showing that human MrgprX3 is activated by hBD3²⁺And (6) tracing. Fig. 20B is a graph of qPCR results showing efficient knockdown of MRGPRX3 from human primary keratinocytes by siRNA. MRGPRX4 was measured as a control. FIG. 20C is a graph demonstrating that a knockout of MRGPRX3 significantly reduces Ca production in response to hBD3²⁺Of human keratinocytesGraph.

Fig. 21A to 21D are a series of immunoblots, the graphs being illustrative showing that murine MrgprA6 is a putative homolog of human MrgprX 3. Fig. 21A is an immunoblot showing that RT-PCR shows high expression of MrgprA6, MrgprA12, and MrgprB3 in purified murine keratinocytes. FIG. 21B is a series of corresponding Ca's showing that murine MrgprA6 is activated by mBD14²⁺And (6) tracing. Fig. 21C is a corresponding tracer indicating that MrgprB3 was not activated by mBD14 (other Mrgpr data not shown). Figure 21D is a schematic showing the Mrgpr gene cluster in the murine and human genomes. The dashed lines indicate the corresponding homologues.

Fig. 22 is a table showing the results of heme metabolites and Mrgpr analysis. Heme metabolites are structurally related. Various heme metabolites activate the murine receptor MrgprA1 and the human receptor MrgprX 4. The dosages of the substances are listed above, but are shown in the table as approximately percent of activation.

Fig. 23A to 23E are images showing Mrgpr-dependent pruritus caused by bilirubin, which is non-histaminergic. Figure 23A is a bar graph indicating the presence of scratching action associated with the injection of bilirubin. The amount of bilirubin noted was injected into the dorsum of the neck of the mice in a volume of 100. mu.L. The blue column (+ HSA) represents animals that have been injected with 60 μ g bilirubin (100 μ L, 1mM) previously incubated with 1% human serum albumin. Vehicle, n ═ 8; 6 μ g, n ═ 5; 18 μ g, n ═ 11; 30 μ g, n-12; 60 μ g, n ═ 7; + HSA, n-12. Fig. 23B is a line graph showing the time course of pruritic behavior associated with injections of bilirubin, histamine, or chloroquine. The scratching action was followed to occur according to 5 minute intervals. Bilirubin, n-16; histamine, n ═ 13; chloroquine, n ═ 11. FIG. 23C is a bar graph showing the results of injecting 60 μ g bilirubin into the back of the neck of WT and cluster-/-littermates. WT, n is 8; cluster-/-, n-13. FIG. 23D is a bar graph showing the results of injecting 60 μ g (100 μ L, 1mM) of the labeled metabolite into WT and cluster-/-littermates. Hemin (WT, n ═ 10; cluster-/-n ═ 6), biliverdin (WT, n ═ 7; cluster-/-n ═ 7), urobilinogen (WT, n ═ 15; cluster KO, n ═ 8), coprolenin (WT, n ═ 7; cluster-/-, n ═ 5). Fig. 23E is a schematic of the heme degradation pathway. The backbone pattern of each metabolite is shown in its optimal 3D geometry as calculated by B3LYP functionality and 6-31g (D) basis set. Blue and orange represent orbital parity of each metabolite HOMO obtained from DFT calculations. Fig. 23A, 23C, and 23D: mean + s.e.m. Each open loop represents one individual mouse. P < 0.05; p < 0.01; p < 0.001; two-tailed unpaired students t-test.

Fig. 24A to 24N are data showing bilirubin activation of murine MRGPRA1 and human MRGPRX 4. FIGS. 24A to 24E are Ca showing HEK293 expression stably of MRGPRA1 or MRGPRX4 (FIGS. 24F to 24J)²⁺Imaging and transformation of the data combined with the isotherms. FIGS. 24A to 24C and FIGS. 24F to 24H are data showing the addition of 50. mu.M bilirubin as indicated by the black bars. After 15 seconds, a1 minute wash was applied. Mean ± 95% Confidence Intervals (CI) are shown. n is 10. In FIG. 24A, 30 μ M FMRF indicated by black bars was added after washing. In FIGS. 24B-24C and 24G-24H, cells were contacted with 10 μ M PLC inhibitor U73122 or 10 μ M G prior to imaging_αqInhibitor YM254890 was incubated for 30 minutes in advance. Bilirubin, conjugated bilirubin, and heme-oriented (FIG. 24D) MRGPRA1, (FIG. 24I) MRGPRX4, and (FIG. 24M) MRGPRC11 and BAM 8-22-oriented MRGPRC11 (a given peptide ligand) -Ca concentrations²⁺Response curves. Data are shown as mean ± s.e.m. in corresponding experiments of 2 to 3 independent replicates performed in triplicate experiments. Bilirubin, conjugated bilirubin, and heme binding isotherms toward (fig. 24E) MRGPRA1, (fig. 24J) MRGPRX4, and (fig. 24N) MRGPRC11 and BAM8-22 to MRGPRC 11. Data are the mean of 3 independent experiments, shown as mean ± s.e.m. Figure 24K shows a histogram of bilirubin-stimulated G protein activity of partially purified MRGPRA1, MRGPRX4, and MRGPRC11 membrane complexes. Measured in the presence of 0.5% DMSO or 50. mu.M bilirubin³⁵S]GTP γ S binding. Shown as mean ± s.e.m. P<0.01; two-tailed unpaired students t-test. FIG. 24L is a bar graph showing the appearance of scratching action in WT and A1-/-animals injected with 60 μ g (100 μ L, 1mM) bilirubin. Shown as the mean plus s.e.m. Open circles represent murine individuals. WT, n is 10; a1-/-, n ═ 12; a, P<0.05, two-tailed unpaired student t test.

FIGS. 25A to 25I show the manifestation of bilirubinData for the activation of sensory neurons by elements in an MRGPR dependent manner. FIG. 25A shows a locus for the endogenous Mrgpra1 gene (Mrgpra 1)^GFP) Images of GFP expression under control of (a). Red shows anti-PLAP antibody staining in which PLAP expression is conferred by endogenous Mrgprd loci (Mrgprd)^PLAP) And (4) controlling. Blue color shows antibody staining against calcitonin gene-related peptide (CGRP). The scale bar is 50. mu.M. FIG. 25B is a graphical representation of whole cell current clamp recordings of WT or A1-/-DRG neurons. In WT DRG, bilirubin elicits action potentials in 5 of 50 small diameter neurons. In A1-/-DRG, bilirubin elicits action potentials of 0of 60 small diameter neurons. Exact probability test P<0.05. FIG. 25C is a representation of images recorded by whole-cell current clamp with 50 μ M bilirubin and 1mM chloroquine (CQ-added WT DRG neurons), respectively, FIG. 25D is a representation of Ca showing WT DRG neurons²⁺A chart of the imaging. After a 10 second baseline, 50 μ M bilirubin was added. After 20 seconds, a3 minute wash was applied before the addition of 1mM chloroquine. After 15 seconds, 50mM KCl was added. Mean plus 95% CI is shown. n is 10 neurons. The applied compound is indicated by black bars. FIG. 25E is a bar graph showing the percent activation of WT, A1-/-and cluster-/-DRG when vehicle or 50 μ M bilirubin was added. A, P<0.05；**，P<0.01；***，P<0.001; chi Square test. FIG. 25F is a graph showing the Tg (Mrgpra3-Cre) assessed by calcium imaging using vehicle, 1mM chloroquine, or 50 μ M bilirubin; lsl-graph of percent activation of tdTomato neurons. If Δ F>0.2 for at least 30 seconds, the neuron is considered to be activated. Fig. 25G to 25H, Ca of cluster-/-DRG neurons and DRG neurons 48 hours after simulated infection with lentivirus (n 10) (fig. 25G) or lentivirus encoding Mrgpra1(n 6), MRGPRX4(n 10) or MRGPRX3(n 20) (fig. 25H)²⁺And (6) imaging. When indicated by a black bar, 50 μ M bilirubin was added. After 20 seconds, a1 minute wash was applied before adding 50mM KCl. The applied compound is indicated by black bars. Mean ± 95% CI is shown. n is 10 neurons. Figure 25I is a graph indicating the percentage of bilirubin-activated cluster-/-neurons uninfected, Mrgpra 1-infected, MRGPRX 4-infected, and MRGPRX 3-infected. P<0.001. Chi Square test.

Fig. 26A to 26I are images showing that Mrgpra 1-/-animals, cluster-/-animals, and BVR-/-animals all exhibit reduced cholestatic itch. FIG. 26A is a bar graph showing the appearance of scratching action in vehicle-treated mice and ANIT-treated mice in WT group, cluster-/-group and A1 KO-/-group. The presence of scratching was assessed over a 30 minute period. For the vehicle cohort: WT, n is 15; cluster-/-, n-6; a1-/-, n ═ 6. For the ANIT queue: WT, n is 20; cluster-/-, n-14; a1-/-, n ═ 14. Fig. 26B is a bar graph showing the appearance of scratching behavior in vehicle treated animals and ANIT treated animals in WT and BVR-/-groups. The presence of scratching was assessed over a 30 minute period. For the vehicle cohort: WT, n is 5; and BVR-/-, n-8. For the ANIT queue: WT, n is 21; and BVR-/-, n ═ 20. Figure 26C is a bar graph showing plasma bilirubin values from ANIT-treated animals and vehicle-treated animals of the WT combination cluster-/-group. For the vehicle cohort: WT, n is 9; cluster-/-, n-5. For the ANIT queue: WT, n is 10; cluster-/-, n-8. Figure 26D is a bar graph showing the appearance of scratching actions in WT ANIT treated animals. Intraperitoneal delivery of vehicle or 1mg/kg QWF. Vehicle, n ═ 8; QWF, n ═ 9. Fig. 26E is a bar graph showing the appearance of scratching action in WT mice injected with vehicle treated plasma or WT animals injected with ANIT treated plasma and BVR-/-animals. For vehicle plasma cohort: for cholestatic ANIT treatment plasma: WT, n is 10; and BVR-/-, n-8. Figure 26F is a table depicting patient characteristics from which hyperbilirubine plasma was collected. FIG. 26G is a bar graph showing scratching behavior in WT mice or A1-/-mice injected with plasma from patients with hyperbilirubinemia. For the cohort injected patient 1 plasma: WT, n is 7; a1-/-, n ═ 9. For the cohort of injected patient 2 plasma: WT, n is 8; a1-/-, n ═ 5. For the cohort injected with patient 3 plasma: WT, n is 7; a1-/-, n ═ 8. For the cohort of injected patient 4 plasma: WT, n is 6; a1-/-, n ═ 8. FIG. 26H is a bar graph showing injection of untreated (NT) control human plasma, FeCl₃Treated control human plasma, NT cholestatic patient 1 plasma (copy of patient 1WT data in FIG. 26G), or FeCl₃Scratch-out of mice of treated patient 1 plasmaNow. For control plasma, NT, n ═ 6; and FeCl₃And n is 5. For patient 1 plasma, NT, n ═ 7; and FeCl₃And n is 7. FIG. 26I is a bar graph showing the appearance of scratching action in mice injected with normal rabbit IgG treated patient 1 plasma or anti-bilirubin IgG treated patient 1 plasma. Normal IgG, n ═ 5; anti-bilirubin, n-7. Fig. 26A to 26I, shown as mean plus s.e.m. The open circles represent individual data points. A, P<0.05；**，P<0.01；***，P<0.001; unpaired two-tailed students t-test.

Figures 27A to 27F show data showing that bilirubin causes non-histaminergic itch but not pain. Fig. 27A is a bar graph showing the presence of scratching action associated with buccal bilirubin injection. A 10 μ l volume of the noted amount of bilirubin was injected and a 30 minute assessment of the presence of scratching action was made. Vehicle, n ═ 6; 1.8 μ g, n ═ 5; 3 μ g, n ═ 4; 6 μ g, n ═ 5; 6 μ g (-/-), n 6. Fig. 27B shows a bar graph, the following rub is associated with injection of 6 μ g bilirubin into the cheek. A 10 minute rub assessment was performed after injection, vehicle, n-5; bilirubin, n-7. FIG. 27B shows a bar graph of the number of licks described below in relation to injection of 6 μ g bilirubin into the cheek. Lick assessment was performed 10 minutes after injection. In each case, n is 3. Fig. 27D shows a bar graph showing that H1 blockers do not inhibit bilirubin-induced pruritus. 30 minutes before the injection of bilirubin in the back of the neck, vehicle or 30mg/kg cetirizine was administered intraperitoneally. Assessment of the appearance of scratching action was performed 30 minutes after injection. Vehicle, n ═ 10; cetirizine, n ═ 5. Figure 27E is a bar graph showing mast cell histamine release in response to 100 μ M bilirubin. The vehicle of compound 48/80, n-4; compound 48/80(10 μ g/mL), n ═ 4; vehicle, n ═ 6; bilirubin, n-8. FIG. 27F is Ca showing murine peritoneal mast cells²⁺A chart of the imaging. After a 10 second baseline, 100 μ M bilirubin was added. After 15 seconds, a1 minute wash was performed, after which 10. mu.g/mL of Compound 48/80 was added. When indicated by black bars, the drug was added. Mean ± 95% CI is shown. n is 26. Fig. 27A to 27C and 27D to 27E, mean + s.e.m. Open circles represent individual data points. A, P<0.05；**，P<0.01；***，P<0.001; two-tailed unpaired students t-test. n.s.: not significant.

FIGS. 28A to 28F are data for bilirubin not activating other MRGPRs described below. Fig. 28A is a graphical illustration of murine and human Mrpgr loci with previously published functional homologous pairs highlighted in black. FIGS. 28B to 28F are Ca showing HEK293 cells transiently expressing MRGPRA3 (FIG. 28B), MRGPRC11 (FIG. 28C), MRGPRD (FIG. 28D), MRGPRX1 (FIG. 28E) or MRGPRX2 (FIG. 28F)²⁺Graph of imaging, if indicated in black bars, 50 μ M bilirubin was added, 15 seconds later, 1 minute wash was applied, after wash, 1mM chloroquine (fig. 28b), 3 μ M BAM8-22 (fig. 28c), 1mM β -alanine (fig. 28d), 3 μ M BAM8-22 (fig. 28E), or 10 μ g/mL compound 48/80 (fig. 28F) were added as indicated in black bars, mean ± 95% ci.n ═ 10.

Fig. 29A to 29C show CRISPR deletion data of MRGPRA 1. FIG. 29A is a schematic showing a comparison of WT and A1-/-genomic sequences. The location of the 2 base pair (bp) deletion is shown in dashed lines. The numbers correspond to the MRGPRA1 open reading frame. FIG. 29B shows sequencing data for a 2bp deletion. FIG. 29C shows a schematic representation of translation of the MRGPRA 1-/-open reading frame starting with the start codon. This 2bp deletion created a frame shift that resulted in premature termination, marked with a red asterisk (extreme right).

Fig. 30A and 30B show images showing bilirubin activation similar to chloroquine in diameter sensory populations. Fig. 30A is a venn diagram showing total neurons activated by bilirubin and/or chloroquine (bilirubin-7 alone, chloroquine-40 alone, overlap-13). Figure 30B is a histogram of bilirubin-activated neuronal cell body diameters.

FIGS. 31A to 31K are graphs showing that there are no differences in plasma levels of the liver injury pathology markers for WT, cluster-/-, A1-/-and BVR-/-animals. Fig. 31A is a graph showing plasma alkaline phosphatase (ALP) levels in animals treated with vehicle and ANIT. For the vehicle cohort: WT, n is 10; cluster-/-, n-4; a1-/-, n ═ 4; BVR-/-, n 6. For the ANIT queue: WT, n is 17; cluster-/-, n-6; a1-/-, n ═ 5; BVR-/-, n 15. Figure 31B is a graph showing plasma aspartate transaminase (AST) levels in vehicle and ANIT treated animals. For the vehicle cohort: WT, n is 10; cluster-/-, n-4; a1-/-, n ═ 4; BVR-/-, n-9. For the ANIT queue: WT, n is 12; cluster-/-, n-6; a1-/-, n ═ 5; BVR-/-, n-17. Figure 31C is a graph showing alanine Aminotransferase (ALT) levels in vehicle and ANIT treated animals. For the vehicle cohort: WT, n is 10; cluster-/-, n-4; a1-/-, n ═ 4; BVR-/-, n 6. For the ANIT queue: WT, n is 15; cluster-/-, n-6; a1-/-, n ═ 5; BVR-/-, n-17. Figure 31D is a graph showing γ -glutamyl transferase (GGT) levels in vehicle and ANIT treated animals. For the vehicle cohort: WT, n is 10; cluster-/-, n-4; BVR-/-, n 6. For the ANIT queue: WT, n is 17; cluster-/-, n-6; BVR-/-, n 15. Fig. 31E is a graph showing plasma bile acid levels (μ M) for ANIT and vehicle treated animals. For the vehicle cohort: WT, n is 4; cluster-/-, n-5; BVR-/-, n-5. For the ANIT queue: WT, n is 10; cluster-/-, n-7; BVR-/-, n-14. Figure 31F is a graph showing methionine enkephalin levels in plasma of vehicle and ANIT treated animals. For the vehicle cohort: WT, n is 4; cluster-/-and BVR-/-, n-5. For the ANIT queue: WT, n ═ 19; cluster-/-, n-10; BVR-/-, n 11. Figure 31G is a graph showing hometown chemokine activity in plasma of vehicle and ANIT treated animals. For the vehicle cohort: WT and BVR-/-, n-4; cluster-/-, n-5. For the ANIT queue: WT, n is 12; cluster-/-, n-8; BVR-/-, n 10. Figure 31H is a graph showing the appearance of scratching action in response to buccal injection of 10 μ L of 1.3mM deoxycholic acid (DCA). WT, n is 9; cluster-/-, n-9. FIG. 31I is a graph showing the appearance of scratching action in response to cheek injection of 10 μ L of 4mM lysophosphatidic acid (LPA). WT, n is 6; cluster-/-, n-6. Fig. 31J shows WT, n-7; cluster-/-, n-8. FIG. 31K is a graph showing the appearance of scratching action in response to a dorsal injection of 25 μ g of DAMGO (50 μ L volume). WT, n is 5; cluster-/-, n-5. Fig. 31A to 31J show the mean ± s.e.m. Open circles represent individual data points. P < 0.05; p < 0.01; p < 0.001; two-tailed unpaired students t-test. n.s.: not significant.

Fig. 32A to 32F are data showing that BVR-/-and a 1-/-animals have a complete itch circuit. FIG. 32A is a bar graph showing quantitative PCR analysis of BLVRA transcripts from WT and BVR-/-murine whole brain. FIG. 32B is a representative chromatogram of an HPLC analysis of plasma from WT and BVR-/-mice, isolated using a C18 column and analyzed by absorption at 450 nm. Figure 32C shows an HPLC chromatogram from WT mouse plasma with excess bilirubin causing a spike. FIG. 32D is a bar graph showing total bilirubin levels of WT and BVR-/-animal plasma. WT, n is 7; BVR-/-, n 6. FIG. 32E is a bar graph showing the appearance of scratching action in response to injection of 150 μ g (50L, 10mM) chloroquine. After chloroquine injection, the appearance of scratching was assessed over a 30 minute period. WT, n is 9; BVR-/-, n-5; a1-/-, n ═ 6. FIG. 32F is a bar graph showing the appearance of scratching action in response to injection of 60 μ g (100 μ L, 1mM) bilirubin. After bilirubin injection, the presence of scratching was assessed over a 30 minute period. WT, n is 8; BVR-/-, n-9. Fig. 32D to 32F show the mean ± s.e.m. Open circles represent individual data points. P <0.01, student t test. n.s.: not significant.

FIGS. 33A and 33B are graphs showing that A1-/-and BVR-/-animals have reduced itching associated with cyclosporin A treatment. Figure 33A is a bar graph showing the appearance of scratching action in WT and a 1-/-animals treated with vehicle and cyclosporin a. For the vehicle cohort: all n-5. For the cyclosporin a cohort: WT, n is 10; and, a1-/-, n-8. Fig. 33B is a bar graph showing the appearance of scratching action in WT and BVR-/-animals treated with vehicle and cyclosporin a. The occurrence of scratching action was assessed over a period of 30 minutes. For the vehicle cohort: n is 5. For the cyclosporin a cohort: WT, n is 11; and BVR-/-, n-7. Fig. 33A and 33B show the mean + s.e.m. The open circles represent individual data points. P < 0.05; p < 0.01; p < 0.001; two-tailed unpaired students t-test. n.s.: not significant.

Figures 34A to 34G show graphs showing that QWF treatment does not affect the severity of cholestatic liver injury. FIG. 34A is bilirubin-induced Ca in HEK cells expressing MRGPRA1²⁺Concentration response plot of signal. 200 μ M bilirubin was maintained in competition with the noted dose of QWF. Mean ± s.e.m. Two parallel experiments were carried out and,each time n is 3. Figures 34B to 34C show histograms showing the appearance of scratching action upon co-injection of 60 μ g (100mL, 1mM) bilirubin (figure 34B) or 150 μ g chloroquine (figure 34C) with vehicle or 1mg/kg QWF. After injection, the number of scratching actions was evaluated over a 30 minute period. For bilirubin: vehicle, n ═ 7; QWF, n ═ 8. For chloroquine: vehicle, n-4; QWF, n ═ 7. Shown as the mean plus s.e.m. P<0.05; unpaired two-tailed students t-test. FIG. 43D values 34G are WT animal plasma bilirubin (FIG. 34D), AST (FIG. 34E), ALT (FIG. 34F) and ALP (FIG. 34G) levels of the dosing vehicle and QWF that have undergone ANIT liver injury. Fig. 34D to 34G show the mean ± s.e.m. Open circles represent individual data points. n.s.: not significant, two-tailed unpaired students t-test.

FIGS. 35A and 35B show data showing FeCl₃And plasma bilirubin depletion by anti-bilirubin antibodies. FIG. 35A shows representative HPLC chromatograms of 100 μ M biliverdin +100 μ M bilirubin standards and treated plasma samples. The absorption was measured at 405 nm. FIG. 35B shows treatment of untreated, FeCl₃Histogram of plasma bilirubin quantification in treated, normal rabbit IgG treated and bilirubin antibody treated samples. Each point represents a technical replication. Mean ± s.e.m. shows ·, P<0.01; n.s., not significant, two-way analysis of variance and subsequent post-hoc Tukey testing.

Detailed Description

The present invention is based, at least in part, on the identification of novel G protein-coupled receptors, human MrgprX4 and murine MrgprA 1. MrgprX4 and MrgprA1 are expressed within specific types of innate immune cells, mediate Stevens-Johnson syndrome (SJS), and appear to be involved in autoimmune disease. MrgprX4 and MrgprA are activated by a variety of drugs that cause SJS including lamotrigine (lamotrigine) and allopurinol (allopurinol). In addition, MrgprX4 and MrgprA are also expressed within sensory neurons and are important for itching sensations and cholestatic pruritus. In some embodiments, MrgprX4 and MrgprA1 are receptors for bilirubin. As described herein, no bilirubin receptor has been identified prior to this finding. In some embodiments, human MrgprX4 is a drug target for SJS, autoimmune diseases such as multiple sclerosis, cholestatic pruritus, and other chronic pruritus. As described herein, prior to this finding, the role MrgprX4 plays in any biological process and disease was completely unknown. In some embodiments, assays based on cells expressing MrgprX 4(MrgprX4 cell line and cDNA, and MrgprA1 mutant murine lines) are used to screen and test drugs targeting these responses. As described herein, the MrgprX 4-expressing cell line is brand new and is used for high throughput screening for drug screening. In some embodiments, blocking MrgprX4 is treatment of SJS; autoimmune diseases such as multiple sclerosis; and novel approaches to cholestatic pruritus and other chronic pruritus.

The present invention is also based, at least in part, on the discovery that human MrgprX3 and its murine congener, MrgprA6, are expressed within primary sensory neurons in keratinocytes, epithelial cells, and Dorsal Root Ganglia (DRGs). Antimicrobial peptide defensins and antimicrobial peptides (cathelicidins) were also found to be agonists of MrgprX3 and MrgprA 6. Defensins and antimicrobial peptides may play a role in a variety of diseases and conditions including wound healing, chronic inflammation, malignant transformation, skin diseases such as psoriasis and dermatitis, respiratory and gastrointestinal diseases, pain and itch. In some embodiments, MrgprX3 and MrgprA6 are targeted for the treatment of wound healing, chronic inflammation, malignant transformation, skin diseases such as psoriasis and dermatitis, respiratory and gastrointestinal disorders, pain and itch. As described herein, the role that MrgprX3 plays in any biological process and disease is completely unknown. In some embodiments, agents targeting these responses are screened and tested using MrgprX 3-based cell-based assays and MrgprA6 mutant mice. As described herein, the MrgprX 3-expressing cell line is novel and useful for high throughput screening for drug screening. The present invention provides methods for determining whether a compound affects a G protein-coupled receptor mediated disorder, and methods for reducing the severity of a G protein-coupled receptor mediated disorder in a subject. The present invention is based, at least in part, on the following findings: g protein-coupled receptors uniquely expressed in immune cells called dendritic cells, MrgprX4 in humans and MrgprA1 in mice, are closely associated with drug side effects and autoimmune diseases.

Prior to the invention described herein, the role played by MrgprX4/MrgprA1 in drug side effects and autoimmune disease was completely unknown. Described herein are methods of screening for drugs that induce drug side effects or autoimmune diseases using assays based on cells expressing MrgprX4/MrgprA1, and screening for MrgprX4 antagonists that block or affect these responses.

The isolated cells of the invention express human G protein-coupled receptor (GPCR) MrgprX4 or murine GPCR MrgprA1, which facilitate visualization of receptor activation in calcium-based screening assays. These cell lines allow screening for MrgprX4 agonist and antagonist activity for FDA approved drugs and drugs under development.

Using these cells for drug screening in cell-based assays, a positive result (i.e., activation of the cell line as measured by, for example, calcium release) would indicate that the drug would normally activate dendritic cells in the patient and potentially cause side effects. Screening of drugs under development will predict their side effects; screening of currently used drugs will identify the causes of the side effects of these drugs; screening for antagonists would lead to new therapeutic drugs that could be provided simultaneously as drugs that induce drug side effects, thus blocking activation of cells (e.g., dendritic cells and primary sensory neurons) without interfering with their intended use.

Cell lines useful for screening FDA-approved drugs and clinical trial compounds that activate or antagonize this receptor are described herein. These will be useful in determining whether a drug will induce an allergic-type response, and in screening to develop antagonists that block these responses.

The introduction of the murine model to study the activation of dendritic cells and primary sensory neurons by drug side effects and the identification of MrgprX4 as a therapeutic target to reduce drug side effects is detailed below.

As described herein, a novel G protein-coupled receptor was identified: human MrgprX4 and murine MrgprA 1. MrgprX4 and MrgprA1 are expressed within specific types of innate immune cells, mediate stevens-johnson syndrome (SJS), and appear to be involved in autoimmune disease. MrgprX4 and MrgprA are activated by a variety of drugs that cause SJS including lamotrigine (lamotrigine) and allopurinol (allopurinol). In addition, MrgprX4 and MrgprA are also expressed within sensory neurons and are important for itching sensations and cholestatic pruritus. MrgprX4 and MrgprA1 are receptors for bilirubin. Prior to this finding, no bilirubin receptor has been identified. Thus, human MrgprX4 is an important drug target. Until this finding as described herein, the role that MrgprX4 plays in any biological process and disease is completely unknown. Therefore, it is entirely new to use assays based on cells expressing MrgprX 4(MrgprX4 cell line and cDNA, and MrgprA1 mutant murine line) to screen for and test drugs targeting these responses. The MrgprX4 expressing cell line is completely new and important for high throughput screening of drug screening. Blocking MrgprX4 may be a novel approach to treating SJS, autoimmune diseases such as multiple sclerosis, as well as cholestatic pruritus and other chronic pruritus.

Targeting MrgprX3 in humans and MrgprA6 in mice can treat wound healing, chronic inflammation, malignant transformation, skin diseases (e.g., psoriasis and dermatitis), respiratory and gastrointestinal disorders, pain and itch, as described herein. Prior to the present disclosure as described herein, the role that MrgprX3 plays in any biological process and disease was unknown. Therefore, it is also novel to use MrgprX 3-expressing cell-based assays and MrgprA6 mutant mice to screen for and test drugs that target these responses. The MrgprX 3-expressing cell line is novel and important for high throughput screening for drug screening.

MrgprX4/MrgprA1

The mass-related G protein-coupled receptor member X4 is a protein encoded by the MRGPRX4 gene in humans. The MAS1 oncogene is a G protein-coupled receptor that binds to the angiotensin-II metabolite angiotensin- (1 to 7). When activated by binding to angiotensin- (1 to 7), the MAS1 receptor has the effect of a wide variety of angiotensin-II activated angiotensin receptors. MAS1 receptor agonists have similar therapeutic effects to angiotensin-II receptor antagonists, including lowering blood pressure.

Drug side reactions (ADR) are a serious unexpected and undesirable drug safety concern that is responsible for approximately 6% of the total hospitalization causes of that and 9% of hospitalization costs, which costs up to $ 301 million per year in the United states alone (Zalewska-Janowska, A., et al, immunological Cleargy Clin North Am 37, 165-. One of the most severe ADRs, Stevens Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis (TEN), are life-threatening severe skin ADRs (cADR), characterized by blister lesions, mucosal rupture and rash/desquamation due to massive keratinocyte death, with mortality rates of up to 30% (Down, A., et al., J Am Acad Dermatol66, 995-. Although involvement of inappropriate immune-mediated cytotoxicity has been shown (downy, a., et al., J Am Acad dermatol66,995-1003(2012)), the molecular and cellular mechanisms how drugs trigger SJS/TEN remain largely unknown. As described herein, a novel drug-induced SJS murine model with ocular mucosal lesions and paw blebs has been established. In some embodiments, several SJS/TEN pathogenic drugs can directly activate G protein-coupled receptor (GPCR) Mrgpra1 and its human functional homolog MRGPRX4 in mice. In Mrgpra1 knockout animals, the drug-induced SJS-like phenotype was abolished. Furthermore, as described herein, Mrgpra1 and MRGPRX4 are both expressed on a subset of dendritic cells that are specialized antigen presenting cells necessary to initiate an adaptive immune response that results in cytotoxicity. Finally, in cADR patients, mutations in the MRGPRX4 gene have been identified that result in increased sensitivity of the receptor to the drug. These findings suggest how these drugs can trigger new molecular mechanisms of severe side effects and open new doors to potential prophylactic and therapeutic measures for ADR.

SJS is a milder form of TEN, first disclosed in 1922 by Albert m.stevens and Frank c.johnson (Stevens, A.M. & Johnson, f.c., Am J Dis child24,526-533 (1922)). Since then, the degree of convulsions to SJS/TEN has increased in the medical field due to its mortality and morbidity. Although several types of infections and malignancies are thought to be implicated in the etiology of SJS/TEN, the major cause of SJS/TEN is a medical side effect (Heng, y.k., et al., Br J Dermatol 173, 1250-. Over 100 clinically used drugs, including antiepileptics (e.g., lamotrigine, carbamazepine), antigout drugs (e.g., allopurinol), antibiotics and nonsteroidal anti-inflammatory drugs for certain tumors have been linked to SJS/TEN, which is a major concern for drug safety and is closely monitored by the Food and Drug Administration (FDA) (Schotland, p., et al, eur J Pharm Sci 94,84-92, (2016)). Immunological responses induced by pathogenic drugs have been considered as the underlying mechanism of SJS/TEN pathogenesis. Although several models have been proposed involving the interaction of pathogenic drugs, Human Leukocyte Antigens (HLA) on antigen presenting cells, and T cell receptors on T cells (Adam, J., et al Br J Clin Pharmacol 71,701-707 (2011); Chung, W.H., et al, J Dermatol 43,758-766(2016)), the molecular and cellular mechanisms of how drugs trigger SJS/TEN remain at least in part a mystery due to the lack of a superior and simple animal model.

Steve Johnson syndrome

Steve Johnson Syndrome (SJS) is a severe form of skin reaction. Together with Toxic Epidermal Necrolysis (TEN), forms a spectrum of diseases, but SJS is less severe. Early symptoms include fever and flu-like symptoms. After some days, the skin begins to blister and slough off, forming the original area of pain. Typically, mucous membranes such as the oral mucosa are also involved. Complications include dehydration, sepsis, pneumonia, and multiple organ failure.

The most common causes are drugs such as lamotrigine, carbamazepine, allopurinol, sulfa antibiotics, and nevirapine (nevirapine). Other causes may include infections such as mycoplasma pneumoniae and cytomegalovirus infections, or the cause may remain unknown. Risk factors include HIV/AIDS and systemic lupus erythematosus. The diagnosis is made based on less than 10% of the skin being implicated. When more than 30% of the skin is involved, it is called TEN, while the intermediate form involves 10% to 30% of the skin. Erythema Multiforme (EM) is generally considered an independent case.

Treatment is typically carried out in a hospital, for example in a burn ward or an intensive care unit. Efforts have included discontinuation of the cause, pain medication, antihistamines, antibiotics, intravenous immunoglobulin, or corticosteroids. Each year, it affects 100 to 200 million people with TEN. Men have twice as many incidence as women. Typically below the age of 30. Skin typically regenerates within two to three weeks, but complete recovery can take months.

Although SJS may be caused by viral infection and malignancy, its major cause is drug. The main reason seems to be the use of antibiotics, especially sulfonamides. SJS may be associated with 100 to 200 different drugs. There is no reliable test to establish a link between a particular drug and an individual case, SJS. The determination of which drug is the cause is made based on the time interval between the first use of the drug and the onset of the skin reaction. A published Algorithm (ALDEN) for assessing drug causality gives a structural aid in identifying reliable drugs.

SJS may be caused by side effects of the following drugs: vancomycin (vancomycin), allopurinol, valproate (valproate), levofloxacin (levofloxacin), diclofenac (diclofenac), etravirine (etravirine), isotretinoin (isotretinoin), fluconazole (fluconazole), valdecoxib (valdecoxib), sitagliptin (sitagliptin), oservir (oseltamivir), penicillin, barbiturate, sulfonamides, phenytoin (phenylytoin), azithromycin (azimycin), oxcarbazepine (oxcarbazepine), zonisamide (zonisamide), modafinil (modafinil), lamotrigine, nevirapine (nevirapyrimethamine), ibuprofen (ibutrofen), ethosuximide (ethosuxim), propiconazole (propiconazole), and felidone (pitazine).

Drugs that are traditionally known to cause SJS, erythema multiforme and toxic epidermal necrolysis include sulfonamides, penicillins, cefixime (antibiotics), barbiturates (sedatives), lamotrigine, phenytoin (e.g., Dilantin) (anticonvulsants), and trimethoprim (trimethoprim). The combination of lamotrigine with sodium valproate increases the risk of SJS.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a rare cause of SJS in adults; the risk is higher for elderly patients, women and initial care givers. Typically, drug-induced symptoms of SJS appear within one week of initial dosing. Like NSAIDs, acetaminophen (acetaminophen) has also caused few SJS cases. People with systemic lupus erythematosus or HIV infection are more susceptible to drug-induced SJS.

Autoimmune diseases

Autoimmune diseases are conditions that arise as a result of an abnormal immune response to a normal body part. There are at least 80 types of autoimmune disease. Almost any body part can be involved. Common symptoms include low fever and feeling tired. The symptoms go forward and backward.

The cause is usually unknown. Some autoimmune diseases, such as lupus, are familial inherited, and some causes may be triggered by infection or other environmental factors. Some common autoimmune diseases include celiac disease, type 1 diabetes, Graves disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus. It may be difficult to confirm the diagnosis.

Treatment depends on the type and severity of the condition. Non-steroidal anti-inflammatory drugs (NSAIDs) and immunosuppressive agents are commonly used. Occasionally intravenous immunoglobulin may also be used. While treatment generally improves symptoms, they typically do not cure the disease.

In the united states, approximately 2,400 million (7%) of people are affected by autoimmune disease. Women have a higher prevalence than men. Onset generally begins in adulthood. The earliest autoimmune diseases were described in the early 1900 s.

The human immune system typically produces T cells and B cells, both of which are reactive with self-antigens, but these autoreactive cells are generally either killed within the immune system before becoming active in an incapacitated state (they remain silenced in the immune system due to over-activation) or they are regulated away from their role in the immune system. When any of these mechanisms fail, there may be a pool of autoreactive cells that become functional in the immune system. The mechanism to prevent autoreactive T cells from being created is performed by a negative selection process within the thymus as the T cells develop into mature immune cells.

Some infections, such as Campylobacter jejuni (Campylobacter jejuni) infections, have antigens that are similar (but not identical) to the host's own autologous cells. In this case, a normal immune response to Campylobacter jejuni can result in the production of antibodies that also react with receptors on skeletal muscle, but to a lesser extent (i.e., myasthenia gravis). One major understanding of the pathophysiological mechanisms of autoimmune diseases is the use of genome-associated scans, which have identified a degree of gene sharing in autoimmune diseases.

On the other hand, autoimmunity is the presence of an auto-reactive immune response (e.g., autoantibodies, autoreactive T cells), with or without damage or pathology resulting therefrom. This may also be restricted to certain organs (e.g. in autoimmune thyroiditis), or involve specific tissues in different locations (e.g. Goodpasture's disease), which may affect basement membranes in both the lung and kidney.

Multiple sclerosis

Multiple Sclerosis (MS) is a demyelinating disease in which the insulating coverings of nerve cells in the brain and spinal cord are damaged. This impairment destroys the ability of nervous system components to communicate, resulting in a range of signs and symptoms, including physical, psychological and sometimes mental problems. Specific symptoms may include double vision, blindness from a single eye, muscle weakness, difficulty in perception, or difficulty in coordination. There are several forms of MS, new symptoms either appearing in isolated episodes (recurrent) or accumulating over time (progressive). Before two attacks, the symptoms can disappear completely; but permanent neurological problems often remain, especially with the development of disease.

Although the cause is unknown, the underlying mechanism is thought to be either destruction by the immune system or failure of myelinating cells. The proposed causes include genetic and environmental factors, e.g. triggered by viral infection. MS is generally diagnosed based on the signs and symptoms present and the results of supporting medical testing.

For multiple sclerosis, there is no known cure. Treatment is attempted after the onset to improve function and prevent new attacks. Drugs used to treat MS, although slightly effective, may have side effects and be poorly tolerated. Physical therapy may contribute to a person's functional mobility. Many people seek alternative treatments, but lack evidence. Long term outcomes are difficult to predict, and good outcomes are more common in women, those who develop the disease early in life, those who have a recurring course of disease, and those who initially experience few episodes. Life expectancy is on average 5 to 10 years shorter than that of the unaffected population.

Multiple sclerosis is the most common autoimmune disease affecting the central nervous system. In 2015, approximately 230 million people worldwide were affected, and the incidence of disease varied widely in different regions and different populations. Approximately 18,900 die in that year from MS, far beyond 12,000 in 1990. The disease typically begins to appear between the ages of 20 and 50, and women have twice as many incidences as men. The name multiple sclerosis refers to the development of a large number of scars (scars-more widely known as plaques or lesions) on the white matter of the brain and spinal cord.

Cholestatic pruritus

Itching (also known as pruritus) is the sensation of a desire or reflex to cause scratching. Itching has resisted many attempts to classify it as any type of sensory experience. Modern science has shown that itch has many similarities to pain, and although both are pleasant sensory experiences, their behavioral response patterns differ. Pain creates an avoidance reflex, while itching causes a scratching reflex. The unmyelinated nerve fibers of the primary sensory neurons in the dorsal root ganglion for itch and pain all originate from the skin, but their information is transmitted centrally in two distinct systems that use the same nerve bundle and spinothalamic tracts.

Cholestatic pruritus is pruritus due to any early liver disease, but the most relevant entities are primary biliary cirrhosis, primary sclerosing cholangitis, obstructive common bile duct stones, bile duct cancer, cholestasis (see also drug-induced pruritus), and chronic hepatitis c virus infection and other forms of viral hepatitis.

Cholestasis means "slow or stagnant bile flow" which may be caused by any number of diseases of the liver (which produces bile), the gall bladder (which stores bile), or the bile duct (also known as the biliary tree, a passage that allows bile to leave the liver and gall bladder and enter the small intestine). When this disease occurs, conjugated bilirubin and waste products, which would normally be cleared in the bile, flow back into the bloodstream. This causes hyperbilirubinemia and jaundice, which are primarily conjugated bilirubin; the liver binds bile to make it water soluble, and because the bile has been processed by the liver, the blood will have a high level of conjugated bilirubin when the bile flows back and back into the blood due to an obstruction. This is in contrast to hyperbilirubinemia, which is predominantly unconjugated bilirubin, which is a water soluble form of bilirubin bound to serum albumin; the liver has no chance of conjugating bilirubin and may either be caused by the formation of too much unconjugated bilirubin (for example in massive haemolysis or ineffective erythropoiesis) or may be caused by too little bilirubin being bound (Gilbert's disease or Crigler-Najjar syndrome). Unconjugated hyperbilirubinemia generally does not cause itching.

It is generally believed that bile salts deposited in the skin are responsible for itching, but the correlation between bilirubin levels in the bloodstream and the severity of itching does not appear to be high. Patients who have been administered bile salt sequestrants do not report some relief, whereas patients who suffer from complete hepatocyte failure (and are therefore unable to produce these as starting products) do not have itching. This suggests that the products produced by the liver must play a role in itch.

Chronic itching or pruritus causes greater distress (Halvorsen JA, et al acta Derm Venereol 92:543-6 (2012)). Clinically relevant chronic pruritus is the result of a variety of pathologies (Ikoma a, et al nat RevNeurosci 7:535-47 (2006)). One major cause is cholestasis. Cholestasis is the result of an impaired ability to secrete bile and can occur due to a variety of pathologies including anatomic obstruction of the bile duct and liver failure (bergasanv. pruritus of cholestasis. in Itch: Mechanisms and Treatment, ed. e cars, takiyama. boca raton (fl) Number of (2014)). Itch caused by cholestasis is non-histaminic and can be addressed using underlying disease pathology solutions (Bergasa nv. prurittus of cholestis. initch: Mechanisms and Treatment, ed. e cardens, T akiyama. boca raton (fl. numberrof. (2014)). It is hypothesized that cholestatic pruritus is caused by the presence of itching-causing agents in the bile. Currently, endogenous opioids, Bile Acids (BA) and lysophosphatidic acid (LPA) are the main candidates for three classes of suggested treatments for cholestatic pruritus.

Endogenous opioids are upregulated in the serum of both cholestatic animal models and patients (Swain MG, et al.1992.gastroenterology 103:630-5 (1992); Thornton JR, Losowsky MS.BMJ 297:1501-4-29 (1988); Thornton JR, Losowsky MS.J. Hepatol 8:53-9 (1989); Thornton JR, Losowsky MS.Gut 30:1392-5 (1989)). In small clinical trials, the opioid antagonists naloxone (naloxone) and nalmefene (nalmefene) proved effective in controlling cholestatic pruritus (Bergasa NV. am JGastroergol 93:1209-10 (1998); Bergasa NV, et al Hepatoloy 27:679-84 (1998); Bergasa NV, et al. gastroenterology 102:544-9 (1992); Swain MG, et al.1992.gastroenterology 103:630-5 (1992)). BA, a steroid metabolite of cholesterol, also increases in serum of cholestatic patients, but their levels are not correlated with the intensity of pruritus reported by the patients (Bergasa NV. pruritussof Cholestasis. in Itch: Mechanisms and Treatment, ed. E Carstens, T Akiyama. bocaRaton (FL). BA-binding resins were shown to be effective in relieving itching in a series of human Clinical trials (Datta DV, Sherlock S.gastroenterology 50:323-32 (1966); European Association for the Study of the L.EASL Clinical practices: management of reactive diseases. J.Hepatol 51:237-67 (2009)). However, these conclusions have been questioned (Kremer AE, et al.gastroenterology 139:1008-18, (2010); Kuiperme, et al.hepatology 52:1334-40 (2010)). In 2013, TGR5, a bile acid receptor, was identified in itch-encoding sensory neurons (Alemi F, et al.J Clin Invest 123:1513-30 (2013)). Finally, homemade chemokines (enzymes that convert lysophosphatidylcholine to LPA) are up-regulated in the serum of cholestatic patients (Kremer AE, et al. This increase occurs uniquely in patients reporting itching (Kremer AE, et al. hepatology 56: 1391-.

Endogenous opioids, BA and LPA are all examples of non-histaminic pruritus. Cholestatic patients do not exhibit traditional signs of histamine release such as erythema or swelling (Bergasa nv. pruritus of cholestasis. in Itch: Mechanisms and Treatment, ed. e stents, T akiyama. boca raton (fl). Furthermore, antihistamines are ineffective in treating cholestatic pruritus, and only a few patients report clinical improvement in their symptoms (Bergasa nv. clin river Dis 12: 385-.

Bile stagnation and bilirubin

Cholestasis will generally lead to jaundice, yellowing of the skin and eyes. Jaundice occurs due to elevated levels of bilirubin deposited in the skin. Bilirubin is a downstream metabolite of heme. Within the cell, heme is cleaved by heme oxidase 1(HMOX1) to biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase (BVR). Bilirubin is extremely lipophilic and is believed to cross cell membranes. In blood, bilirubin is bound by albumin. In the liver, UGT1A × 28 binds bilirubin to glucuronic acid to form water soluble compounds. Both conjugated and unconjugated bilirubin is excreted in the bile. In terms of human health, bilirubin is believed to be a physiological antioxidant with cardiovascular protective benefits (Bulmer AC, et al. prog Lipid Res 52:193-205 (2013); Vitek L., et al. Atheroschesis 160:449-56 (2002)).

Mrgpr and non-histaminergic pruritus

Mas-related G-protein coupled receptors (Mrgpr) have been implicated in non-histaminergic pruritus as receptors for pro-itchy ligands and as molecular markers of pruritus-encoding neurons (Liu Q, et al. J Neurosci 32:14532-7 (2012); Liu Q, et al. cell 139:1353-65 (2009)). There are over 27 Mrgpr expressed in mice, only a portion of which has a known physiological ligand (McNeil B, Dong x. neurosci Bull 28:100-10 (2012)). There are 4 Mrgpr expressed in humans (MrgprX1 to MrgprX 4). MrgprX1, MrgprX3 and MrgprX4 have been identified as being specifically expressed in human DRG and Trigeminal Ganglia (TG), while MrgprX2 has been found in human mast cells (Flegel C, et al. PLoS One 10: e0128951 (2015); Goswami SC, et al. mol Pain 10:44 (2014); Lembo PM, et al. Nat Neurosci 5:201-9 (2002); McNeil BD, et al. Nature 519:237-41 (2015)).

MrgprX3/MrgprA6

The mass-related G protein-coupled receptor member X3 is a protein encoded by the MRGPRX3 gene in humans. The MAS1 oncogene is a G protein-coupled receptor that binds to the angiotensin-II metabolite angiotensin- (1 to 7). When activated by binding to angiotensin- (1 to 7), the MAS1 receptor has the effect of a wide variety of angiotensin-II activated angiotensin receptors. MAS1 receptor agonists have similar therapeutic effects to angiotensin-II receptor antagonists, including lowering blood pressure.

Human MrgprX3 and its murine homolog MrgprA6 are expressed in primary sensory neurons within keratinocytes and Dorsal Root Ganglia (DRGs). More importantly, the antimicrobial peptides defensins and antimicrobial peptides (cathelicidins) are agonists of MrgprX3 and MrgprA 6. Defensins and antimicrobial peptides may play a role in a variety of diseases and conditions including wound healing, chronic inflammation, malignant transformation, skin diseases such as psoriasis and dermatitis, respiratory and gastrointestinal diseases, pain and itch. Targeting MrgprX3 and MrgprA6 can treat wound healing, chronic inflammation, malignant transformation, skin disorders such as psoriasis and dermatitis, respiratory and gastrointestinal disorders, pain and itch. Prior to the present disclosure as described herein, the role that MrgprX3 plays in any biological process and disease was unknown. Therefore, it is also novel to use MrgprX 3-expressing cell-based assays and MrgprA6 mutant mice to screen for and test drugs that target these responses. The MrgprX 3-expressing cell line is novel and important for high throughput screening for drug screening.

Injury and pathogen invasion trigger a cascade of inflammatory and repair responses directed to the restoration of damaged tissue. It has also long been noted that repeated irritation and chronic inflammation are strong risk factors for cancer. A thorough understanding of the wound healing process will therefore provide an important insight into the causes of various cancers and, accordingly, its prevention. The bulk of the host defense molecule released during this process is a large family of antimicrobial peptides (AMPs) known as defensins. These AMPs are particularly interesting as they exert a homeostatic immunomodulatory effect on various types of cells at various stages of the inflammatory response in addition to directly killing pathogens. In particular, the human β -defensin hBD3 has been shown to aid in wound healing by stimulating epithelial cell migration and proliferation. As described herein, a novel G protein-coupled receptor (GPCR) -MRGPRX 3-is a defensin receptor in human keratinocytes and other types of epithelial cells. As described herein, the ligand homology and expression profiles for the murine gene MrgprA6, which is a murine homolog of human MRGPRX3, as well as the physiological and cellular biological functions of these receptors were examined in vitro and in vivo.

The skin is the largest immune organ and the first line of defense against infectious challenges. The inflammatory cascade is rapidly triggered as a protective response when injury and pathogen invade (Paspalakis, M., et al. Nat RevImmunol 14, 289-301 (2014); Singer, A.J. & Clark, R.A.F.N.Engl.J.Med.341, 738-746 (1999)). Local skin inflammation is characterized by "redness with heat and pain," as described by ancient romans (Owen, j.a., et al., immunology. (w.h. freeman,2013)), and involves multiple types of cells and a number of molecular mediators. This complex process involves a concerted action of the immune, nervous, vascular and epithelial systems and is crucial for our survival.

Human β -defensin hBD3 promotes keratinocyte migration and wound healing defensins are a large family of antimicrobial peptides (AMPs) that are produced by epithelial and immune cells immediately following tissue damage and infection and can kill the spectrum of pathogens (Pazgier, M., et al. cell. mol. Life Sci.C.63, 1294-1313 (2006); Amid, C.et al. BMC Genomics 10, 1-13 (2009); defensins also exert a variety of immune regulatory functions in addition to direct killing, human β -defensin 3(hBD3) exhibit a wide range of functions, including chemotactic activity for various immune cells (Ganz, T.Nat Rev Immunol 3, 710-720 (2003);j, et al.j.immunol.184, 6688-6694 (2010), mast cell degranulation (Befus, a.d.et al.j.immunol.163, 947-953 (1999); subramanian, h.et al.j.immunol.191, 345-352 (2013)), and has been shown to aid in wound healing (Hirsch, t.et al.j.gene med.11, 220-228 (2009);o.e.et al.j.immunol.170, 5583-5589 (2003); aarbbiu, J.et al.am.J.Respir.cell mol.biol.30, 193-201 (2004); otte, j. -m.et al.j.cell.biochem.104, 2286-2297 (2008)). Experiments with primary human keratinocytes in culture showed that hBD3 promotes keratinocyte migration and proliferation (Niyonsaba, F.et al.J.invest.Dermatol.127, 594-604 (2016)). When applied to infected diabetic wounds, hBD3 significantly reduced bacterial load and promoted epidermal cell regeneration and wound closure. The effect of hBD3 on keratinocytes was blocked by pertussis toxin, indicating that the receptor was a GPCR (Niyonsaba, F.et al.J.invest.Dermatol.127, 594-604 (2016)).

MRGPRX3 is the major hBD3 receptor in human keratinocytes. MRGPRX3 belongs to the Mas-related G-protein coupled receptor (mrgprr) family of GPCRs. As described herein, studies over the past decade have revealed different expression patterns and functions of these receptors in sensory neurons and immune cells (Liu, Q.et al. cell 139, 1353-1365 (2009); Han, L.et al. Nat Neurosci 16, 174-182 (2013); McNeil, B.D.et al. Nature 519,237-241(2015)). Of the 4 human MRGPRX, MRGPRX3 is highly expressed in epithelial cells including keratinocytes, while the others are specific for DRG neurons, mast cells or other immune cell types (hrruz, t.et al. adv. bioinformatics 2008,420747 (2008); kitsurayanon, c.et al. j.dermotol. sci. doi: http:// dx. doi.org/10.1016/j.jdermsci. (2016.05.006)). Keratinocyte dysplasia was induced in rats by overexpression of human MRGPRX3 using a universal promoter (Kaisho, y.et al. biochem. biophysis. res. commun.330, 653-657 (2005)). Ca in Primary human keratinocytes as described herein²⁺Imaging and knock-out experiments demonstrated that MRGPRX3 is required for cells to respond to hBD 3. Expression analysis and ligand homology further indicate that the murine gene MrgprA6 acts as a murine homologue of MRGPRX3, opening the door to in vivo studies of these receptors.

Wound healing

In intact skin, the epidermis (superficial layer) and dermis (deeper layer) form a protective barrier against the external environment. When the barrier is broken, a carefully designed set of biochemical cascades are set to initiate to repair the damage. This process is divided into predictable stages: blood clotting (hemostasis), inflammation, tissue growth (proliferation), and tissue remodeling (suppuration). Blood coagulation can be considered as part of the inflammatory phase rather than as a separate phase.

Hemostasis (blood clotting) is the beginning of the early stages of the wound healing process. Within the first few minutes after injury, platelets in the blood begin to stick to the injured site. This activates platelets, causing something to happen. They change to an indeterminate shape that is more suitable for coagulation, and they release a chemical signal to promote coagulation. This results in the activation of fibrin, which forms a network and acts as a "glue" to bind the platelets to each other. This forms a clot that acts to embolize the vascular rupture, slowing/preventing further bleeding.

During the validation period, damaged and dead cells are cleared along with bacteria and other pathogens or debris. This occurs through a process of phagocytosis in which white blood cells "eat" the debris by engulfming it. During the proliferation phase, platelet-derived growth factors are released into the wound, causing migration and division of cells.

During the proliferative (new tissue growth) phase, collagen deposition, granulation tissue formation, epithelialization and wound contraction occur. In angiogenesis, vascular endothelial cells form new blood vessels. In fibrous and granulation tissue formation, fibroblasts grow and form a new, temporary extracellular matrix (ECM) by excluding collagen and fibronectin. At the same time, epidermal cell regeneration of the epidermis occurs, wherein the epithelial cells proliferate and "crawl" over the wound surface, providing coverage of new tissue. In wound contraction, myofibroblasts reduce wound size by grasping the wound edges and contract using a mechanism similar to that in smooth muscle cells. As the role of the cell approaches completion, unwanted cells undergo apoptosis.

During maturation and remodeling, collagen realigns along the tension lines, removing cells that are no longer needed through programmed cell death or apoptosis. The wound healing process is not only complex but also fragile, and it is easily interrupted or failed, resulting in the formation of non-healing chronic wounds. Factors responsible for non-healing chronic wounds are diabetes, venous or arterial disease, and senile metabolic defects.

Dendritic cells

Dendritic Cells (DCs) are antigen presenting cells (also called helper cells) of the mammalian immune system. Their main function is to process antigenic material and present it on the surface of T cells of the immune system. They act as messengers between the innate immune system and the adaptive immune system. Dendritic cells are present in those tissues that are in contact with the external environment, such as the skin, where selected from the specific dendritic cell types known as Langerhans cells, and the inner walls of the nose, lungs, stomach, and intestines. They may also be found in the blood in an immature state. Once activated, they migrate to the lymph nodes where they interact with T and B cells to initiate and shape adaptive immune responses. Immature dendritic cells are also called veil cells because they possess large cytoplasmic "veil" rather than dendrites.

Dorsal root ganglion

The dorsal root ganglion (or spinal ganglion), also known as the dorsal root ganglion, is a cluster of nerve cell bodies (ganglia) in the dorsal root of the spinal nerve. The dorsal root ganglion contains the cell bodies of sensory neurons (afferents). Sensory neurons, also known as afferent neurons, are neurons that convert a specific type of stimulus into action or hierarchical potentials via their receptors. This process is called sensory transduction. The cell bodies of sensory neurons are localized in the dorsal ganglia of the spinal cord. The primary sensory neuron is the first of the afferent pathways, starting at the receptor and terminating at a synapse with a secondary sensory neuron, typically within the core of the central nervous system.

This sensory information travels along afferent fibers, within afferent or sensory nerves, through the spinal cord to the brain. The stimulation may be from external receptors outside the body, such as light and sound, or from internal receptors within the body, such as blood pressure or postural sensation. Different types of sensory neurons have different sensory receptors that respond to different kinds of stimuli.

Axons of dorsal root ganglion neurons are also known as afferent nerves. In the peripheral nervous system, afferent nerves refer to axons that transmit sensory information to the central nervous system (i.e., the brain and spinal cord). Neurons are composed of three parts: a dendron that receives the information and delivers it to a somatic cell; somatic cells, the cell bodies of neurons; and axons, which convey information from the autologous cells. In a neuron, a dendrite receives information from the axon of another neuron at the synapse, and the axon sends information to the dendrite of the next neuron, even though the dendrite may be covered with myelin.

Proton-sensing G protein-coupled receptors are expressed by dorsal root ganglion sensory neurons and may play a role in acid-induced nociception. In some embodiments, G protein-coupled receptors (e.g., MrgprX4 or MrpgrA1) in the primary sensory neurons of the dorsal root ganglion mediate sensations such as pain and itch.

The nerve endings of dorsal root ganglion neurons have various sensory receptors that are activated by mechanical stimuli, thermal stimuli, chemical stimuli, and noxious stimuli. The high threshold channel may play a role in nociception. Presynaptic modulation of dorsal nerve endings released in the spinal cord may occur through certain types of GABAA receptors, which may control nociception and pain transmission.

HEK293 cell

Human embryonic kidney 293 cells, also commonly known as HEK293, HEK-293, 293 cells, or less precisely HEK cells, are derived from human embryonic kidney cells grown in tissue culture (from aborted human embryos) and from specific cell lines of stillborn animals. HEK293 cells are very growth-competent, highly transfectable, and have been widely used for many years in cell biology research. They are also used in the biotechnology industry to produce therapeutic proteins and viruses for gene therapy. HEK293 cells stably expressing MrgprX3 or MrgprX4 are described herein.

Pharmaceutical composition

In certain embodiments, the invention provides pharmaceutical compositions comprising the agents employed in the invention. The agent can be stably formulated and introduced into the subject's body or cellular environment by any means known for such delivery.

Such compositions typically comprise the agent and a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial and antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or glucose. The pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be sealed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble), or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor el.tm. (BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that syringability is readily achieved. It should be stable under the conditions of manufacture and storage and must be protected against contaminating action by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization which result in a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions typically include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compounds can be combined with excipients and used in the form of tablets, dragees, or capsules, such as gelatin capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following or a compound of similar character: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose; disintegrating agents, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or a fragrance such as peppermint, methyl salicylate, or orange flavoring.

The compositions of the present invention may also be formulated as nanoparticle formulations. The compounds of the present invention may be administered for intermediate release, delayed release, modified release, sustained release, pulsed release and/or controlled release applications. The pharmaceutical composition of the present invention may contain 0.01% to 99% (w/v) of the active material. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or from a nebulizer. Such methods include those described in U.S. patent 6,468,798.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal summary, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be performed through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated in ointments, sleeves, gels or creams, as is generally known in the administration art. The compounds may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compound is prepared with a carrier that will protect the compound from rapid clearance from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Such formulations may be prepared using standard techniques. Materials are commercially available from Alza Corporation (Alza Corporation) and nova pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells and monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. This can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD₅₀(dose lethal to 50% of the population) and ED₅₀(50% of the population is a therapeutically effective dose). The dosage ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as LD₅₀/ED₅₀And (4) the ratio. Compounds exhibiting a high therapeutic index are preferred. Although compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to unaffected cells, thereby reducing side effects.

Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dose of such compounds is preferably in the circulating concentration range, which includes the low or non-toxic ED₅₀. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For compounds used in the methods of the invention, a therapeutically effective dose can be initially evaluated from cell culture assays. The dose can be formulated in animal models to achieve a circulating plasma concentration range, including as measured in cell cultureThe obtained IC₅₀(i.e., the concentration of test compound that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine the dose available in a human. For example, levels in plasma can be measured by high performance liquid chromatography.

As defined herein, a therapeutically effective amount (i.e., effective dose) of an agent depends on the agent selected. For example, a single dose of agent in the range of about 1pg (picogram) to 1000mg may be administered; in some embodiments, 10pg, 30pg, 100pg, 1000pg, 10ng, 30ng, 100ng, 1000ng, 10 μ g, 30 μ g, 100 μ g, 1000 μ g, 10mg, 30mg, 100mg, 1000mg can be administered. In some embodiments, 1 to 5g of the composition can be administered.

A therapeutically effective amount of a compound of the invention can be determined by methods known in the art. The therapeutically effective amount of the pharmaceutical composition of the invention depends, in addition to the selected agent and/or the pharmaceutical composition used, on the age and general physiological condition of the patient and on the route of administration. In certain embodiments, the therapeutic dose will generally be between about 10 and 2000 mg/day, and preferably between about 30 and 1500 mg/day. Other ranges may be used, including, for example, 50 to 500 mg/day, 50 to 300 mg/day, 100 to 200 mg/day.

Administration may be once daily, twice daily, or more frequently, and may be less frequent during the maintenance phase of the disease or disorder, e.g., once every two or three days rather than once daily or twice daily. The dosage and frequency of administration will depend on clinical signs which confirm the presence of a remission period with a reduction or absence of at least one or more, preferably more than one, acute phase clinical sign known to those skilled in the art. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, prior treatments, the general health and/or age of the subject, and other diseases present. Furthermore, treatment of a subject with a therapeutically effective amount of an agent may comprise a monotherapy, or, optionally, may comprise a series of therapies.

It will be appreciated that the method of introducing the agent into the environment of the cell will depend on the type of cell and the composition of its microenvironment. Appropriate amounts of the agent must be introduced and these amounts can be determined empirically using standard methods. Exemplary effective concentrations of the individual agents in the cellular environment can be 500 micromoles/liter or less, 50 micromoles/liter or less,10 micromoles/liter or less,1 micromoles/liter or less, 500 nanomoles/liter or less, 50 nanomoles/liter or less,10 nanomoles/liter or less, or even compositions having concentrations of 1 nanomole/liter or less can be used.

The pharmaceutical composition may be included in a kit, container, package or dispenser with instructions for administration.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the assays, screens, and therapeutic methods of the invention can be made and used, and are not intended to limit the scope of what the inventors regard as their invention.