Systems, Models and Methods for Identifying and Evaluating Skin-Active Agents Effective for Treating Dandruff/Seborrheic Dermatitis

• US 20120258074A1
• Filed: 04/02/2012
• Published: 10/11/2012
• Est. Priority Date: 03/31/2011
• Status: Active Grant

• Current Assignee: The Procter & Gamble Company
• Original Assignee: The Procter & Gamble Company
• Inventors: MILLS, KEVIN JOHN, BINDER, ROBERT LLOYD, YOUNGQUIST, ROBERT SCOTT, XU, JUN, HU, PING, TAMURA, MAKIO
• Granted Patent: US 10,072,293 B2
• Time in Patent Office: Days
• US Class Current: 424/85.2
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/148 Screening for cosmetic compounds
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Methods and systems for determining functional relationships between a skin-active agent and a skin condition of interest, and methods and systems for identifying cosmetic agents effective for treatment of dandruff, as well as the use of agents identified by such methods and systems for the preparation of cosmetic compositions, personal care products, or both are provided. Methods for developing in vitro models of skin disease and models for specific skin diseases are also provided.

Claims

1. A method for constructing a data architecture for use in identifying connections between perturbagens and genes associated with dandruff, comprising:

(a) providing a gene expression profile for a control human epidermal keratinocyte cell;

(b) generating a gene expression profile for a human epidermal keratinocyte cell exposed to at least one perturbagen;

(c) identifying genes differentially expressed in response to the at least one perturbagen by comparing the gene expression profiles of (a) and (b);

(d) creating an ordered list comprising identifiers representing the differentially expressed genes, wherein the identifiers are ordered according to the differential expression of the genes;

(e) storing the ordered list as a keratinocyte instance on at least one computer readable medium; and

(f) constructing a data architecture of stored keratinocyte instances by repeating (a) through (e), wherein the at least one perturbagen of step (a) is different qualitatively or quantitatively for each keratinocyte instance.

2. A method according to claim 1, comprising using a programmable computer to perform one or more of steps (c), (d), (e) and (f).

3. A method according to claim 1, wherein the ordered list comprises the ordered list of identifiers in association with a numerical ranking for the identifier corresponding to its rank in the ordered list.

4. A method according to claim 1, wherein the step of generating is performed by extracting a biological sample from the treated cell and subjecting the biological sample to microarray analysis.

5. A method according to claim 1, wherein the biological sample comprises mRNA.

6. A method according to claim 1, wherein the microarray is a global microarray or a specific microarray, wherein the specific microarray comprises oligonucleotides which hybridize to genes corresponding to a gene expression signature for a cellular phenotype.

7. A method according to claim 1, wherein the step of constructing the data architecture of stored instances by repeating steps (a) through (e) comprises repeating steps (a) through (e) for between about 50 and about 50,000 instances.

8. A method according to claim 7, wherein the step of constructing a gene expression data bases of stored instances comprises repeating steps (a) through (e) for between about 1000 and about 20,000 instances.

9. A method according to claim 1, wherein the at least one perturbagen is an anti-dandruff agent.

10. A method according to claim 9, wherein the anti-dandruff agent induces a host response to produce a host effect, or induces anti-fungal activity to produce an anti-fungal effect, or both.

11. The method according to claim 10, wherein the anti-dandruff agent induces a host response to produce a host effect.

12. The method according to claim 11, wherein the host response is restoration of epidermal homeostasis present in healthy scalp skin.

13. The method according to claim 12, wherein restoration of epidermal homeostasis is assessed by measuring a shift in a transcriptional profile derived from scalp skin of the host toward a transcriptional profile of healthy scalp skin.

14. A method according to claim 1, wherein the identifiers are selected from the group consisting of gene names, gene symbols, microarray probe set ID values, and combinations thereof.

15. A method according to claim 1, wherein the ordered list is arranged so that an identifier associated with a most up-regulated gene is positioned at the top of the ordered list and an identifier associated with a most down-regulated gene is positioned at the bottom of the ordered list.

16. A method according to claim 15, wherein the ordered list of each instance is arranged so that an identifier associated with each gene that is not differentially expressed is positioned between the identifier associated with the most up-regulated gene and the identifier associated with the most down-regulated gene.

17. A method according to claim 1, wherein each instance comprises between about 1,000 and about 50,000 identifiers.

18. A method according to claim 1, wherein each instance comprises metadata for the at least one perturbagen associated with the instance.

19. A method according to claim 1, wherein at least one perturbagen is an anti-fungal agent.

20. A method according to claim 19, wherein an anti-fungal agent comprises zinc pyrithione (ZPT), selenium sulfide or both.

21. A method according to claim 1, wherein at least one perturbagen comprises an environmental stimuli.

22. The method according to claim 11 wherein the host response comprises one or more of inducing lipid metabolism, suppressing inflammation, suppressing cell proliferation, suppressing cell apoptosis and normalizing cell differentiation.

23. The method according to claim 22, wherein the host response comprises inducing lipid metabolism and suppressing inflammation.

24. A method for implementing the data architecture according to claim 1 to generate connections useful for identifying skin-active agents effective for treating dandruff, the method comprising querying the data architecture with a dandruff gene expression signature, wherein querying comprises comparing the dandruff gene expression signature to each stored keratinocyte instance, wherein the dandruff expression signature represents genes differentially expressed in association with the condition.

25. A method according to claim 24, wherein the comparison of the dandruff gene expression signature to each stored keratinocyte instance is performed by a programmable computer.

26. A method according to claim 24, wherein the dandruff condition results from a host predisposition, a disruption in epidermal homeostasis in scalp skin, a fungus present in scalp skin, or combinations thereof.

27. A method according to claim 24, wherein the dandruff expression signature is constructed by a method comprising (i) identifying genes having up-regulated expression in the dandruff condition when compared to a control;

(ii) identifying genes having down-regulated expression in the dandruff condition when compared to a control;

(iii) creating one or more gene expression signature lists associated with the dandruff gene expression signature comprising identifiers corresponding to a plurality of the genes identified in (i) and (ii); and

storing the one or more gene expression signature lists on the at least one computer readable medium.

28. A method according to claim 27, wherein the number of genes having up-regulated expression in the dandruff condition is between about 10 and about 400, and the number of genes down-regulated in the dandruff condition is between about 10 and about 400.

29. A method according to claim 28 wherein the identifiers for from between about 80% and about 100% of the up-regulated genes are set forth as in Table A and wherein identifiers for from between about 80% and about 100% of the down-regulated genes are set forth in Table B.

30. A method according to claim 24, wherein the identifiers representing the genes identified in (i) and (ii) are selected from the group consisting of gene names, gene symbols, and microarray probe set ID values.

31. A method according to claim 27, wherein the one or more gene expression signature lists comprises a first list representing a plurality of the up-regulated genes identified in (i) and a second list representing a plurality of down-regulated genes identified in (ii).

32. A method according to claim 27, wherein at least one skin sample is taken from a human subject exhibiting the dandruff condition, a biological sample is extracted from the skin sample, and a gene expression profile of the at least one skin sample is generated prior to at least one of the steps (i) and (ii).

33. A method according to claim 24, wherein the comparison further comprises assigning a connectivity score to each of a plurality of instances.

34. A method according to claim 33, wherein a plurality of connectivity scores represents a positive correlation and a plurality of the connectivity scores represents a negative correlation.

35. A method according to claim 33, wherein the connectivity score has a value between +2 and −

2.

74. A method for identifying a skin-active agent having predictable efficacy in treatment of a skin condition, the method comprising:

a. determining a gene expression signature for the skin condition wherein the gene expression signature comprises genes significantly up- and down-regulated in skin sampled from a subject affected with the skin condition when compared to skin sampled from a subject not affected with the skin condition;

b. determining a thematic expression signature for the skin condition by mapping the genetic expression signature on a biological processes grid to determine one or more regulated processes, wherein a theme expression signature reflects the most regulated processes;

c. providing a connectivity map data architecture according to claim 1;

d. querying the connectivity map with the thematic expression signature determined in (b) to generate an output of skin-active agents;

e. rank-ordering the output by connectivity score wherein a negative connectivity score predicts efficacy of a skin-active agent for the treatment of the skin condition.

75. The method according to claim 74 wherein the skin condition is dandruff.

76. The method according to claim 75 wherein the thematic expression signature includes genes from a dandruff gene expression signature statistically clustered in biological process themes, wherein the biological process themes are selected from the group consisting of lipid metabolism, inflammation, cell proliferation, cell apoptosis and cell differentiation.

77. The method according to claim 76 wherein the biological process themes consist of lipid metabolism and inflammation.

78. The method according to claim 77 wherein a skin-active agent is predicted to have efficacy for the treatment of dandruff where it exhibits negative connectivity to a down-regulation in lipid metabolism and an up-regulation in inflammation.

36. A method for formulating an anti-dandruff composition by identifying connections between perturbagens and genes associated with a dandruff condition, comprising:

(a) accessing a plurality of instances stored on at least one computer readable medium, wherein each instance is associated with a perturbagen and a skin cell type and wherein each instance comprises an ordered list comprising a plurality of identifiers representing a plurality of up-regulated and a plurality of down regulated genes;

(b) accessing at least one dandruff gene expression signature stored on the at least one computer readable medium, wherein the dandruff gene expression signature comprises one or more lists comprising a plurality of identifiers representing a plurality of up-regulated genes and a plurality of down-regulated genes associated with a dandruff condition;

(c) comparing the dandruff gene expression signature to the plurality of the instances, wherein the comparison comprises comparing each identifier in the one or more gene expression signature lists with the position of the same identifier in the ordered lists for each of the plurality of instances;

(d) assigning a connectivity score to each of the plurality of instances; and

(e) formulating an anti-dandruff composition comprising a dermatologically acceptable carrier and at least one perturbagen, wherein the connectivity score of the instance associated with the at least one perturbagen has a negative correlation.

37. A method according to claim 36, further comprising applying the anti-dandruff composition to a plurality of human subjects having a dandruff condition.

38. A method according to claim 37, wherein the anti-dandruff composition reduces flaking and/or itching in one or more of the plurality of human subjects.

39. A method according to claim 36, wherein the identifiers are selected from the group consisting of gene names, gene symbols, and microarray probe set ID values.

40. A method according to claim 36, wherein each instance comprises between about 50 and about 400 identifiers.

41. A method according to claim 36, wherein the plurality of instances comprises between about 50 and about 50,000 instances.

42. A method according to claim 36, wherein the plurality of instances comprises between about 1000 and about 20,000 instances.

43. A method according to claim 36, wherein the at least one perturbagen is an anti-inflammatory agent.

44. A method according to claim 36, wherein the at least one perturbagen promotes lipid metabolism.

45. A method according to claim 36, wherein the at least one perturbagen is I117, I122 or both I117 and I122.

46. A method according to claim 36 wherein one or more of steps (a), (b), (c), and (d) are performed by a programmable computer.

47. A method according to claim 36, wherein the dandruff gene expression signature comprises a plurality of dandruff gene expression signatures and each of the plurality of instances has a connectivity score assigned thereto for each of the plurality of dandruff gene expression signatures.

48. A method according to claim 47, wherein the connectivity score for each of the plurality of instances is a combination of the connectivity scores assigned to each instance for each of the plurality of dandruff gene expression signatures.

49. A method according to claim 48, wherein each of the plurality of dandruff gene expression signatures comprises one or more gene expression signature lists comprising a plurality of identifiers representing a plurality of up-regulated genes and a plurality of down-regulated genes, wherein an identifier for between about 80% and about 100% of the up-regulated genes are set forth in Table A and wherein an identifier for between about 80% and about 100% of the down-regulated genes are set forth in Table B.

50. A method according to claim 48, wherein each connectivity score assigned to the instance associated with the at least one perturbagen of the anti-dandruff composition has a negative correlation.

51. A method according to claim 36, wherein the plurality of instances are stored in a database on the at least one computer readable medium.

52. A method according to claim 51, wherein each of the plurality of instances further comprises metadata associated with the skin cell type and the perturbagen associated therewith.

53. A method according to claim 52, wherein the metadata comprises a name for the skin cell type and a name for the perturbagen.

54. A method according to claim 36, wherein the plurality of instances is stored in a first digital file and the dandruff gene expression signature is stored in a second digital file.

55. An anti-dandruff composition formulated according to the method of claim 36.

56. A method for generating a gene expression signature for use in identifying connections between perturbagens and genes associated with a dandruff condition, the method comprising:

(a) providing a gene expression profile for a reference sample of human scalp skin cells;

(b) generating a gene expression profile for at least one sample of human scalp skin cells from a subject exhibiting a dandruff condition,

(c) comparing the expression profiles of (a) and (b) to determine a gene expression signature comprising a set of genes differentially expressed in (a) and (b);

(d) assigning an identifier to each gene constituting the gene expression signature and ordering the identifiers according to the direction of differential expression to create one or more gene expression signature lists;

(e) storing the one or more gene expression signature lists on at least one computer readable medium.

57. The method according to claim 56, wherein the human skin cells derive from an epidermal layer of scalp skin.

58. A gene expression signature determined according to claim 56 having from about 20 to about 400 genes differentially up-regulated in the dandruff condition and about 20 to about 400 differentially down-regulated in the dandruff condition.

59. A method according to claim 56, wherein the identifiers are selected from the group consisting of gene names, gene symbols, and microarray probe set IDs.

60. A method according to claim 56, wherein the at least one sample of human scalp skin cells comprises a plurality of samples and wherein one of the plurality of skin samples is taken from a dandruff-involved location, one of the plurality of skin samples is taken from a dandruff-uninvolved location, and one of the plurality of skin samples is taken from a non-dandruff subject.

61. A system for identifying connections between perturbagens and genes associated with a dandruff condition, comprising:

(a) at least one computer readable medium having stored thereon a plurality of instances, and a dandruff gene expression signature, wherein the instances and the gene expression signature are derived from a human epidermal keratinocyte cell, wherein each instance comprises an instance list of rank-ordered identifiers of differentially expressed genes, and wherein the dandruff gene expression signature comprises one or more gene expression signature lists of identifiers representing differentially expressed genes associated with a dandruff condition;

(b) a programmable computer comprising computer-readable instructions that cause the programmable computer to execute one or more of the following;

(i) accessing the plurality of instances and the dandruff gene expression signature stored on the computer readable medium;

(ii) comparing the dandruff gene expression signature to the plurality of the instances, wherein the comparison comprises comparing each identifier in the gene expression signature list with the position of the same identifier in the instance list for each of the plurality of instances; and

(iii) assigning a connectivity score to each of the plurality of instances.

62. A system according to claim 61, further comprising:

a microarray scanner for receiving a sample comprising human epidermal keratinocyte cells; and

a second programmable computer for transmitting gene expression data from the scanner to the first programmable computer.

63. A system according to claim 62, further comprising an array of perturbagens for application to the epidermal keratinocyte cells.

64. A system according to claim 63, wherein the plurality of instances comprises between about 50 and about 50,000 instances.

65. A system according to claim 64, wherein the plurality of instances comprises between about 1,000 and about 20,000 instances.

66. A gene expression signature for dandruff consisting of genes selected from the genes set forth in Tables A and B.

67. An immobilized array of oligonucleotides which hybridize to the genes selected for the gene expression signature according to claim 66.

68. A gene expression signature according to claim 66 stored on a memory device accessible by a programmable computer.

69. A gene expression signature according to claim 66, comprising from 20-300 genes identified to be up-regulated in Table A.

70. A gene expression signature according to claim 66, comprising from 20-300 genes identified to be down-regulated in Table B.

71. A gene expression signature according to claim 66, comprising a set of genes identified to be up-regulated and a set of genes identified to be down-regulated.

72. A computer readable medium, comprising:

a data architecture comprising a digital file stored in a spreadsheet file format, a word processing file format, or a database file format suitable to be read by a respective spreadsheet, word processing, or database computer program, the first digital file comprising data arranged to provide one or more gene expression signature lists comprising a plurality of identifiers when read by the respective spreadsheet, word processing, or database computer program; and

wherein each identifier is selected from the group consisting of a microarray probe set ID, a human gene name, a human gene symbol, and combinations thereof representing a gene set forth in any of Tables A-B wherein each of the one or more gene expression signature lists comprises between about 50 and about 600 identifiers.

73. The computer readable medium of claim 72, further comprising computer readable instructions for reading the digital file.

79. A method for producing an in vitro model of a skin disease useful for evaluating clinical efficacy of proposed therapeutic agents in treatment of the skin disease, the method comprising:

a. determining a gene expression signature for the disease state wherein the gene expression signature comprises genes significantly up and down regulated in the disease;

b. conducting a biological process analysis of the regulated genes to identify biological processes implicated by the regulated genes;

c. treating a skin culture to simulate the biological processes identified in (b)

d. confirming validity of the in vitro model of the skin disease by determining the gene expression signature for the treated skin culture and assessing the degree to which it mimics the gene signature determined in (a).

80. A method for producing an in vitro model for a skin disease according to claim 79, wherein the skin disease comprises a chronic keratinous condition.

81. The method according to claim 80, wherein the chronic keratinous condition is selected from dandruff, psoriasis, acne and atopic dermatitis.

82. An in vitro skin model for dandruff comprising a skin culture treated with IL17 and IL22, wherein a gene transcription analysis of the treated skin culture substantially mimics a gene transcription analysis of skin exhibiting dandruff.