Method for Predicting Skin Sensitizing Activity of Compounds
The present invention provides methods for predicting the in vivo skin sensitizing activity of chemical compounds using a combination of mammalian cell models with multiple endpoint analysis, time and concentration response curves. The methods allow the determination of a predicted in vivo sensitization value of a compound—for example, a EC3 LLNA value, a GPMT value or a IVTI value—without the use of animals, with a high degree of accuracy. The methods involve detecting expression levels of genes implicated in skin sensitization, combining expression level data with concentration response data, conducting a computational analysis, and comparing test compound data to a database of known skin sensitizers.
1 . A method for predicting the in vivo skin sensitizing activity of a compound, comprising:
(a) culturing mammalian cells;
(b) applying a concentration of a test compound to the cells of step (a);
(c) measuring the expression level of one or more marker genes in the cells of step (b);
(d) optionally monitoring multiple endpoints of cell viability and general cell health;
(e) conducting a computational analysis of the concentration applied in step (b) and the expression level(s) measured in step (c); and
(f) determining a predicted in vivo sensitization value based on the analysis of step (e).
2 . The method of claim 1 , further comprising plotting the value calculated in step (e) against one or more known LLNA EC3 values and determining a predicted LLNA EC3 value.
3 . The method of claim 1 , wherein the mammalian cells are selected from the group consisting of: human keratinocytes (HaCat cells), 3D human skin cells, normal human epithelial cells (NHEK cells), MCF7 cells, H4IIE cells, and combination cultures including keratinocyte and dendritic cells.
4 . The method of claim 1 , wherein the test compound is applied to the cells in varying dosage amounts.
5 . The method of claim 1 , wherein the marker gene(s) are selected from the group consisting of: quinone reductase, IL-8, ALDHc, CYP1A, GCLA, GST, HO1, MafF, NQO1, hMTT, GAPDH CD-86, AKR, TXN, and TXN reductase.
6 . The method of claim 1 , wherein the in vivo sensitization value is a LLNA EC3 value.
7 . The method of claim 1 , wherein the in vivo sensitization value is a GPMT value.
8 . The method of claim 1 , wherein the in vivo sensitization value is a IVTI value.
9 . The method of claim 1 , further comprising performing the following steps prior to performing step (a):
(i) incubating the test compound with a polypeptide to allow for binding; and
(ii) measuring the amount of unbound polypeptide.
10 . The method of claim 9 , wherein the polypeptide is GSH.
11 . The method of claim 9 , wherein the test compound of step (i) is applied in varying dosage amounts.
12 . The method of claim 9 , further comprising adding human liver microsomal protein in step (i).
13 . A method for determining the sensitizing activity of a compound comprising:
(a) incubating a test compound with a polypeptide to allow for binding; and
(b) measuring the amount of unbound polypeptide.
14 . The method of claim 13 , wherein the polypeptide is GSH.
15 . The method of claim 13 , wherein the test compound is applied in varying dosage amounts.
16 . The method of claim 13 , further comprising adding human liver microsomal protein in step (a).