Tofacitinib (CP-690550) is a potent, small molecule inhibitor of JAK-3, which exhibiting potent effects in preclinical transplantation and arthritis models. It displays greater antiproliferative and pro-apoptotic activity against murine multipotent factor-dependent cell Patersen-erythropoietin receptor (FDCP-EpoR) cells harboring JAK2(V617F) compared with JAK2(WT). JAK-3 has been shown to play a key role in cytokine signaling via gammac, e.g. IL-2, 4, 7, 9, 15, 21. In vitro, Tofacitinib effectively inhibited a murine mixed lymphocyte reaction (MLR) (IC50= 91 nm). Mice chronically dosed with CP-690550 (1.5-15 mg/kg/day) demonstrated dose- and time-dependent alterations in lymphocyte subsets when examined by flow cytometry. The most dramatic change observed was a 96% reduction in splenic NK1.1 + TCRbeta- cell numbers following 21 days of treatment. Delayed-type hypersensitivity (DTH) responses in sensitized mice were reduced in a dose-dependent manner following treatment with the JAK-3 inhibitor (1.87-30 mg/kg, s.c.). Extended survival of neonatal Balb/c hearts implanted into the ear pinna of MHC mismatched C3H/HEN mice was observed with CP-690550 monotherapy (10-30 mg/kg/day), but improved upon combination with cyclosporin (10 mg/kg/day). Furthermore, the ability of Tofacitinib to extend cardiac allograft survival in murine models suggests it may afford a new treatment for prevention of transplant rejection.
Global transcriptional analysis of whole-skin biopsies showed that both drugs also blocked the dermal inflammatory signature, as measured by Alopecia Areata Disease Activity Index
Application
grafted C3H/HeJ mice
Results
we observed complete hair regrowth within 12 weeks following topical therapy. Topical therapy was associated with a markedly reduced proportion of CD8+NKG2D+ T cells in the treated skin and lymph node, normalization of the ALADIN transcriptional signature, reversal of histological markers of disease.
Application
Flow cytometric analysis, Immunohistochemistry and immunofluorescence
Results
we systemically administered tofacitinib at the time of grafting and found that they prevented the development of AA and the expansion of CD8+NKG2D+ T cells in all grafted recipients. The skin of mice treated with either drug showed no histological signs of inflammation. Global transcriptional analysis of whole-skin biopsies showed that both drugs also blocked the dermal inflammatory signature, as measured by Alopecia Areata Disease Activity Index.
Application
Flow cytometric analysis, Immunohistochemistry and immunofluorescence
Results
"We observed complete hair regrowth within 12 weeks following topical therapy. Topical therapy
was associated with a markedly reduced proportion of CD8+NKG2D+T cells in the treated skin and lymph node, normalization of the ALADIN transcriptional signature, reversal of histological markers of disease. "
Application
Fourmice were treated with control (half of the back skin) and ruxolitinib (half of the back skin) and four mice were treated with control (half of the back skin) and tofacitinib (half of the back skin).
Results
Indeed, ~90% of 8.5-week-old mice treated with ruxolitinib or tofacitinib for 5 days displayed skin darkening and hair growth within 10 days of starting treatment, whereas no hair growth was evident in control-treated mice (P < 0.0001 for ruxolitinib treatment and P = 0.04 for tofacitinib treatment). Ruxolitinib treatment enriched for the mTOR (mammalian target of rapamycin) and NFkB (nuclear factor kB) pathways, both previously shown to be involved in hair cycle regulation, whereas tofacitinib treatment enriched for pathways involved in cell motility and migration, such as Rho and integrin signaling.
Application
Human HF organ culture assay
Results
We found that treatment with JAK inhibitors significantly increased the length of hair shafts when treatedwith ruxolitinib and tofacitinib, indicating a positive effect on the rate of hair elongation (P = 0.023 and P = 0.025 for tofacitinib and ruxolitinib, respectively).
Application
Patch assay with DP spheres
Results
In the region including genes thatwere upregulated by ruxolitinib treatment but down-regulated by tofacitinib treatment, we identified proapoptotic genes such as BAX, BCL2L11, and CASP12 (region 2).
Application
HE stain
Results
"At the end of the experiment, mostly anagen hairs were observed in the tofacitinib-treated group, whereas mostly catagen and anagen hairs were observed in minoxidil- and DMSO-treated groups."
Application
HE stain
Results
More newly formed capillaries were observed in the tofacitinib-treated group than in the minoxidil- and controltreated groups, although some completely formed capillaries were observed in the minoxidil- and DMSO-treated groups.
Application
HE stain
Results
Tofacitinib-treated mice showed less infiltration of inflammatory cells relative to minoxidil-treated mice. In the DMSO-treated group, infiltration of several inflammatory cells infiltration was observed, while fewer inflammatory cells were observed in the ethanol-treated group.
Application
Inject
Results
We examined the effect of topical tofacitinib on hair growth in mice tissue, tofacitinib-treated group has shown denser in thickness and length of hair follicle compared to vehicle (Figure 1A). Histology, tofacitinib treated group has shown increased in anagen hair follicle compared to vehicle (Figure 1B).
Application
qRT-PCR
Results
We investigate the expression of BMP4 which known to be an inhibitory molecule for onset of anagen entry. Quantitative real-time reverse transcription-PCR analysis showed decrease expression of BMP4 in tofacitinib treated group compare to DMSO-treated group (p-value 0.003).
Application
oral
Results
None of the treated mice developed alopecia areata or cutaneous lymphadenopathy, whereas untreated mice manifested both AA and associated cutaneous lymphadenopathy
Application
i.v.
Results
In mice treated topically with tofacitinib, 84.7% of hairs were in the anagen phase (n=72), 10.6% were in the catagen phase (n = 9), and 4.7% were in the telogen phase (n=4), where the follicles in the late anagen were located in the deep dermis and hypodermis, and follicles in the early anagen were located in the superficial dermis.
Application
i.v.
Results
The length of the hair infundibulum on the 21st day of the experiment was higher in groups treated topically with tofacitinib.
Protocol (for reference only)
Cell Experiment
Cell lines
cytokine-dependent NK92 cell line
Preparation method
(A) After cytokine starvation for 24 hours, NK92 cells were stimulated by the addition of human IL-2 for 48 hours with and without the addition of serially increasing concentrations of tofacitinib. 3H-thymidine was added during the last 6 hours of the cultures. Cells were then harvested and analyzed for 3H-thymidine incorporation. (B) Cytokine-starved NK92 cells were stimulated with human IL-2, combined IL-6/IL- 6R, or IL-12 for 48 hours with and without serially increasing concentrations of tofacitinib. (C) The NK92 cells were treated as those in panel B with and without a single 50nM dose of tofacitinib. Data are presented as means ±SD (A-C) and are representative of 3 independent experiments. (D) After cytokine starvation for 24 hours, NK92 cells were stimulated with 30 ng/mL of IL-2, 100 ng/mL of combined IL-6/IL-6R, or 100 ng/mL of IL-12 for 1 hour with and without the addition of tofacitinib. The cell lysates were immunoblotted with an anti–phospho-STAT5 monoclonal antibody and an anti-STAT5 antibody. ß-Actin was used as an input control. Data are representative of 3 independent experiments.
Concentrations
0~400 nM
Incubation time
48 h
Animal Experiment
Animal models
IL-15–transgenic CD8 T-cell leukemia–bearing mice
Formulation
dissolved in polyethylene glycol 300 (PEG300; VWR Scientific Products)
Dosages
50 mg/mL
Administration
continuously administered via a subcutaneous mini-osmotic pump (ALZET)
Ruxolitinib (INCB018424) is a first-in-class, potent, selective JAK1/2 inhibitor with an IC50 of 3.3 nM/2.8 nM. It is more than 130-fold more selective for JAK1, JAK2, and JAK3 than JAK3. It can be used in studies related to pemphigus, myelofibrosis and vitiligo.
NVP-BSK805 dihydrochloride is a potent and selective quinoxaline JAK2 inhibitor with IC50 values of 0.48, 0.56 and 0.58 nM for JAK2 JH1, FL JAK V617F and FL JAK2 wt respectively.
Baricitinib (INCB28050) is an oral JAK1 and JAK2 inhibitor with IC50 of 5.9 nM and 5.7 nM, respectively. Baricitinib has potential anti-inflammatory, immunomodulatory and anti-tumor activity.
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