Tofacitinib

Biological Activity

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.

Product Citations

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  EMBO Mol Med. 2022 Aug 8;14(8):e15653.

  Single‐cell transcriptomics reveals a senescence‐associated IL‐6/CCR6 axis driving radiodermatitis
  Tofacitinib purchased from AbMole

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  Front Immunol. 2022 May 23;13:866638.

  A Novel STAT3 Gain-of-Function Mutation in Fatal Infancy-Onset Interstitial Lung Disease
  Tofacitinib purchased from AbMole

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  Front Immunol. 2021 Jul 29;12:675542.

  Tofacitinib Ameliorates Lupus Through Suppression of T Cell Activation Mediated by TGF-Beta Type I Receptor
  Tofacitinib purchased from AbMole

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  J Virol. 2020 Feb 14;94(5):e01791-19.

  Targeting Kaposi's Sarcoma-Associated Herpesvirus ORF21 Tyrosine Kinase and Viral Lytic Reactivation by Tyrosine Kinase Inhibitors Approved for Clinical Use
  Tofacitinib purchased from AbMole

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  Cell Stem Cell. 2019 Apr 4;24(4):654-669.e6.

  A Subset of TREM2+ Dermal Macrophages Secretes Oncostatin M to Maintain Hair Follicle Stem Cell Quiescence and Inhibit Hair Growth.
  Tofacitinib purchased from AbMole

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  Arch Dermatol Res. 2018 Oct 5.

  Effect of tofacitinib on the expression of noggin/BMP-4 and hair growth stimulation in mice
  Tofacitinib purchased from AbMole

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  TJPS. 2017;41 (Supplement Issue): 225-228.

  Topical tofacitinib reduce expression of BMP-4 in mouse hair follicle
  Tofacitinib purchased from AbMole

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  Arch Dermatol Res. 2017 Sep 7.

  Efficacy of topical tofacitinib in promoting hair growt in non‑scarring alopecia: possible mechanism via VEGF induction
  Tofacitinib purchased from AbMole

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  Sci Adv. 2015 Oct 23.

  Pharmacologic inhibition of JAK-STAT signaling promotes hair growth.
  Tofacitinib purchased from AbMole

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  Nat Med. 2014 Sep;20(9):1043-9.

  Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.
  Tofacitinib purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Archives of Dermatological Research (2018). Figure 2. Tofacitinib (Abmole Bioscience, Houston, USA)
	Method	i.v.
	Cell Lines	C57BL/6 mice
	Concentrations	concentration of 2%
	Incubation Time	21 consecutive days
	Results	The length of the hair infundibulum on the 21st day of the experiment was higher in groups treated topically with tofacitinib.

	Source	Archives of Dermatological Research (2018). Figure 1. Tofacitinib (Abmole Bioscience, Houston, USA)
	Method	i.v.
	Cell Lines	C57BL/6 mice
	Concentrations	concentration of 2%
	Incubation Time	21 consecutive days
	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.

	Source	US Patent 9730877B2 (2017). Figure 32. Tofacitinib (Abmole Bioscience)
	Method	oral
	Cell Lines	mice
	Concentrations	15 mg/kg/day
	Incubation Time	12 weeks
	Results	None of the treated mice developed alopecia areata or cutaneous lymphadenopathy, whereas untreated mice manifested both AA and associated cutaneous lymphadenopathy

	Source	TJPS (2017). Figure 2. Tofacitinib (Abmole Bioscience, Houston, TX, USA)
	Method	qRT-PCR
	Cell Lines	mice hair follicle
	Concentrations	2% concentration
	Incubation Time	21 days
	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).

	Source	TJPS (2017). Figure 1. Tofacitinib (Abmole Bioscience, Houston, TX, USA)
	Method	Inject
	Cell Lines	C57BL/6 male mice
	Concentrations	2% concentration
	Incubation Time	21 days
	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).

	Source	Arch Dermatol Res (2017). Figure 6. Tofacitinib (Abmole Bioscience, Texas, USA)
	Method	HE stain
	Cell Lines	Mice
	Concentrations	dissolved in DMSO to create a 2% concentration
	Incubation Time	21 day
	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.

	Source	Arch Dermatol Res (2017). Figure 5. Tofacitinib (Abmole Bioscience, Texas, USA)
	Method	HE stain
	Cell Lines	Mice
	Concentrations	dissolved in DMSO to create a 2% concentration
	Incubation Time	21 day
	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.

	Source	Arch Dermatol Res (2017). Figure 4. Tofacitinib (Abmole Bioscience, Texas, USA)
	Method	HE stain
	Cell Lines	Mice
	Concentrations	dissolved in DMSO to create a 2% concentration
	Incubation Time	21 day
	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."

	Source	Science Advance (2015). Ruxolitinib, Figure 4. (AbMole Bioscience)
	Method	Patch assay with DP spheres
	Cell Lines	DP cells
	Concentrations	400 nM
	Incubation Time
	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).

	Source	Science Advance (2015). Ruxolitinib, Figure 3. (AbMole Bioscience)
	Method	Human HF organ culture assay
	Cell Lines	human HFs
	Concentrations	400 nM
	Incubation Time
	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).

	Source	Science Advance (2015). Ruxolitinib, Figure 1. (AbMole Bioscience)
	Method	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).
	Cell Lines
	Concentrations
	Incubation Time
	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.

	Source	Nature medicine (2014). Figure 1. tofacitinib (Abmole)
	Method	Flow cytometric analysis, Immunohistochemistry and immunofluorescence
	Cell Lines
	Concentrations	15 mg/kg/day
	Incubation Time	12 weeks
	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. "

	Source	Nature medicine (2014). Figure 3. tofacitinib (Abmole)
	Method	Flow cytometric analysis, Immunohistochemistry and immunofluorescence
	Cell Lines
	Concentrations	15 mg/kg/day
	Incubation Time	12 weeks
	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.

	Source	Nature medicine (2014). Figure 1. JAK3i tofacitinib (Abmole)
	Method	grafted C3H/HeJ mice
	Cell Lines	CD8+NKG2D+ T cells
	Concentrations	15 mg/kg/day
	Incubation Time	12 weeks
	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.

	Source	Nature medicine (2014). Figure 1. JAK3i tofacitinib (Abmole)
	Method	grafted C3H/HeJ mice
	Cell Lines	CD8+NKG2D+ T cells
	Concentrations	15 mg/kg/day
	Incubation Time	12 weeks
	Results	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

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)

Chemical Information

Molecular Weight	312.37
Formula	C16H20N6O
CAS Number	477600-75-2
Solubility (25°C)	DMSO ≥60 mg/mL
Storage	-20°C, sealed

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Papp et al. Br J Dermatol. Efficacy and safety of tofacitinib, an oral Janus kinase inhibitor, in the treatment of psoriasis: a Phase 2b randomized placebo-controlled dose-ranging study.

[2] de Lartigue J et al. Drugs Today (Barc). Tofacitinib for the treatment of moderate to severe rheumatoid arthritis.

[3] Labranche et al. Arthritis Rheum. JAK inhibition with tofacitinib suppresses arthritic joint structural damage through decreased RANKL production.

[4] Sandborn et al. N Engl J Med. Tofacitinib, an oral Janus kinase inhibitor, in active ulcerative colitis.