Page 267 - ebook

Intracellular Delivery Of Nuclear Localization Signal Alleviates
Atopic Dermatitis By Regulating The Inflammatory Skin Environment
Jaehyeon Kim, Seokwon Lee, Hyunji Lee, Hyeontae Kang, Mingu Kang, Eunhee Yang,
Eunna Chung and Daewoong Jo
Cellivery R&D Institute, Cellivery Therapeutics, Inc., Seoul 03929, Korea.
BACKGROUND
Atopic dermatitis (AD), which occurs on the skin, is a serious inflammatory autoimmune disease that many people suffer from. Production of
pro-inflammatory cytokines/chemokines is regulated by a certain type of inflammation-associated transcription factors (IAFTs) such as NF-κB,
STAT1/3, NFAT, and AP-1. Improved Cell-Permeable nuclear import inhibitor (iCP-NI) has been developed by fusing a cyclically-linked NLS of
IATFs with advanced macromolecule transduction domain (aMTD), a sequence optimized hydrophobic cell-penetrating peptide (CPP). iCP-NI
suppresses the nuclear transport of IRTFs by competitively binding to importin, reducing pro-inflammatory cytokines.
AIM METHODS
The aim of this study is to develop an iCP-NI as a Nuclear fraction assay revealed suppressed nuclear translocation of IATFs after
therapeutic molecule for autoimmune atopic iCP-NI treatment. The therapeutic effect of iCP-NI cream (w/v, 5%) has been
dermatitis by determining its mode of action and verified with a dinitrochlorobenzene (DNCB)-induced AD mouse model. The
efficacy on regulating expression of pro-inflammatory cream-formulated iCP-NI was topically applied, twice a day, then expressions of
cytokines. inflammatory cytokines and histology of skin barrier were analyzed.
RESULTS

Figure 1. Improved Cell-Permeable Figure 2. Cell And Mouse Skin Figure 5. iCP-NI Recovery Skin Lesion Of Atopic Dermatitis
Nuclear Import Inhibitor (iCP-NI) Dermal Layer Delivery Of iCP-NI
Structure Of iCP-NI Cell Permeability (Keratinocyte) Scoring Atopic Dermatitis (SCORAD) *p<0.05, **p<0.01
HaCaT Cell DNCB 15 Error Bar: SD *
R K
Q R (20 μM, 1 H) No Treatment - Cream Only iCP-NI Cream
V Q Non-CP-NI iCP-NI
C
K 10
C L 50%
P M Scoring Atopic Dermatitis (SCORAD)
aMTD cNLS Cy5-Conjugated 5
• 3 rd Generation Hydrophobic • Nuclear Localization Sequence
Cell Penetrating Peptide (CPP) (NLS) Of NF-κB 10 μm
0
No - Cream iCP-NI
Treatment Only Cream
Dermal Transduction DNCB
Atopic Dermatitis Skin Lesion Skin Thickness 160 *p<0.05, **p<0.01
(25 mg/kg, 2 H, Cream, Topical) Error Bar: SD
DNCB 120
No Treatment - Cream Only iCP-NI Cream 39 % *
Non-CP-NI Epidermal Thickness (μm) 80 68%
Cy5-Conjugated Scale bar = 200 μm 40
No
iCP-NI 5 μm 0 Treatment - Cream iCP-NI
Cream
Only
CD4+ T Helper Cell DNCB
20 μm *p<0.05, **p<0.01
300 Error Bar: SD *
DNCB
No Treatment - Cream Only iCP-NI Cream
Figure 3. iCP-NI Inhibits Nuclear Import Of IATFs In Human Keratinocyte (HaCaT Cell) 200
150
Atopic Dermatitis Related TARC Secretion In Conditioned Media Helper T Cells Number 250 95%
Signaling Pathways 100
Nucleus Cytoplasm *p<0.05, **p<0.01
800 Error Bar: SD Scale bar = 50 μm 50
TNF-α /IFN-γ - + + + - + + + **
0
iCP-NI (μM) - - 1 10 - - 1 10 No - Cream iCP-NI
p-p65 p-p65 Mast Cell Treatment Only Cream
600
p-STAT6 p65 37% DNCB 150 *p<0.05, **p<0.01 DNCB
GATA3 GAPDH Error Bar: SD *
Lamin B1 Human TARC (pg/ml) 400 No Treatment - Cream Only iCP-NI Cream 120
TNF-α + IFN-γ 90 66%
No Treatment - iCP-NI Mast Cells Number
200 60
NF-κB (p-p65) 0 Scale bar = 100 μm 30
No
iCP-NI
Treatment - iCP-NI (10 uM) 0 No - Cream iCP-NI
(1 uM)
Treatment Only Cream
Scale Bar = 10 μm TNF-α / IFN-γ (10 ng/ml)
DNCB
Figure 4. iCP-NI Reduces IgE Levels In Plasma And Pro-Inflammatory Cytokines CONCLUSION
IgE In Plasma Inflammatory Cytokines In Skin Tissue These results suggest that iCP-NI restored AD-like skin lesion
*p<0.05, **p<0.01
10000.0 Error Bar: SD TNF-α IL-6 (50%), as assessed by SCORAD, and the levels of serum IgE (71%),
** 2.5 3 *p<0.05, **p<0.01 6 *p<0.05, **p<0.01 * and iCP-NI reduced the upregulated cytokines level such as IL-6
Total IgE in Plasma (ng/mL) 6000.0 55% 68% TNF-α (pg/ml) 1.5 2 * IL-6 (pg/ml) 5 4 3 (66%), TNF-α (87%). In conclusion, these results suggest that iCP-
8000.0
*
Error Bar: SD
Error Bar: SD
NI has a significant therapeutic effect on AD, an inflammatory
4000.0
skin disease, through immune system control.
2000.0
0.5 1 87% 2 1 66%
0.0 REFERENCES
No - Cream 2% 5% 10% 0
Treatment Only No Cream iCP-NI 0 No Cream iCP-NI
iCP-NI Cream - -
Treatment Only Cream Treatment Only Cream Chung et al. (2020) Science Advances
DNCB
DNCB DNCB
Lim et al. (2013) Clinical Cancer Research
Filaggrin Expression (In Vivo)
TNF-α/IFN-γ Lim et al. (2013) Biomaterials
No DNCB-Induced AD Model
iCP-NI
Treatment - iCP-NI 48 (kDa) - - 1 10 (μM) Contact Information
Filaggrin Epidermis Filaggrin
35 (kDa) Dongho Kim, Cellivery Therapeutics, Inc.
α-tubulin
kimdh@cellivery.com, +82-2-3151-8900
Scale Bar = 100 μm S4-3 신진과학자