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Hydrophobic Cell Penetrating Peptide-Combined
Nuclear Localization Signal Suppresses Pro-Inflammatory Cytokines
In Inflammatory Bowel Disease
Seokwon Lee, Jaehyeon Kim, Hyunji Lee, Hyeontae Kang, Mingu Kang, Eunhee Yang, Eunna Chung and Daewoong Jo
Cellivery R&D Institute, Cellivery Therapeutics, Inc., Seoul 03929, Korea.
BACKGROUND
Inflammatory bowel disease (IBD) includes two types of inflammatory diseases, ulcerative colitis (UC) and Crohn’s disease (CD). A
hydrophobic cell-penetrating peptide-combined nuclear localization signal (NLS), named improved Cell-Permeable nuclear import inhibitor
(iCP-NI), was used to target the IBD. The NLS of iCP-NI was adopted from NF-κB and cyclized for in vivo stability enhancement. iCP-NI
competitively binds to the importins, the nuclear transporter, and suppresses the nuclear import of transcription factors upregulating the
cytokines.
AIM METHODS
This study aims to develop an iCP-NI as a The efficacy of iCP-NI against IBD was evaluated in the DSS-induced UC mouse model and
therapeutic molecule for IBD by determining TNBS-induced CD mouse model. After the mouse was diseased, iCP-NI was intravenously
its mode of action and efficacy in regulating (IV) or intrarectally (IR) administered. The scoring indexes were used to estimate the
the expression of pro-inflammatory cytokines. microscopic or histological analysis. The cytokine expressions were also evaluated.
RESULTS

Figure 1. The Structure And Mode Of Action (MoA) Of Figure 3. Therapeutic Effect Of iCP-NI
Improved Cell-Permeable Nuclear Import Inhibitor (iCP-NI) On DSS-Induced Ulcerative Colitis Mouse Model
Cytoplasm iCP-NI Treatment (50 mg/kg, IV Injection)
Inflammation Activated Once A Day, 7 Days
Transcription Factors (IATFs)
Initiating Factors NF-κB NFAT C57BL/6 Day 0 … 6 7 … 9 … 13
Microbial (Female, 8 Weeks)
Genetic Factors Environmental STAT AP-1
Factors Dysbiosis 3% DSS In Drinking Water, 7 Days Sampling Sampling Endoscopy & Sampling
Antibiotics iCP-NI
Immune Cell Recruiting
Food Intake Importin α
Smoking Competitive H&E Staining : Intestinal Structure
Activation Of Inflammatory Cascades
Inhibition
Upregulating Pro-Inflammatory Cytokines Nucleus Colon Length 3% DSS
(i.e. IL-6, IL-12, IL-18, IL-23, IL-1β, And TNF-α)
Impaired Epithelial Barrier Functions IATFs No Treatment Diluent iCP-NI
Importin α Importin α
Development Of IBD
No Treatment
Figure 2. Restored Expression Of E-Cadherin In Colonic Epithelial Cell Line 3% DSS Diluent
Treated With Dextran Sulfate Sodium (DSS) iCP-NI I.V Day 7
Scale Bar: 200 μm
Immuno-Fluorescence Labeling : α-E-Cadherin 10 Day 7
Endoscopy
Western Blot : α-E-Cadherin 2% DSS 8 * P<0.05 3% DSS
Control Diluent iCP-NI 31% No Treatment Diluent iCP-NI
2% DSS 6
iCP-NI (μM) - - 1 10 50 Colon Length (Cm)
100 4
E-Cadherin
75 E-Cadherin / DAPI 2
50
β-actin
38 0 No Diluent iCP-NI (IV)
Treatment 3% DSS
Day 13
Colonic Epithelial Cell Line (Caco-2) Scale bar = 20 μm
Figure 4. Therapeutic Effect Of iCP-NI On 2, 4, 6-Trinitrobenzenesulfonic Figure 5. Downregulated Cytokine Level In The Plasma Of
Acid Solution (TNBS)-Induced Crohn’s Disease (CD) Mouse Model TNBS-Induced CD Mouse Model By iCP-NI Treatment
iCP-NI Treatment (50 mg/kg)
IV or IR Injection, Twice A Day CBA Assay : Expression Of Pro-Inflammatory Cytokines
BALB/c Day 1 2 3 4 5 6 ** P=0.0001 30 ** P=0.007
(Male, 8 Weeks) * ** P=0.78 P=0.007 ** P=0.0017 ** P=0.0003
TNBS (25 mg/kg in 40% EtOH) Sampling 4 5 4 25 20
Pathological Score 3 3 15
9 TNF-α Concentration (pg/ml) 2 IFN-γ Concentration (pg/ml) IL-6 Concentration (pg/ml)
Colon Length * * P=0.001 64% 2 10
* * P=0.001 1 1 78% 5 82% 72%
Pathological Score (0~9)
No Treatment 6 0 No - IV IR 0 No - IV 97% 0 No - IV IR
IR
Diluent Treatment iCP-NI Treatment iCP-NI Treatment iCP-NI
IV 3 65% 71% TNBS TNBS TNBS
iCP-NI
IR CONCLUSION
0
No - IV IR
Treatment iCP-NI
TNBS The expression of E-cadherin, the junction protein, was restored with iCP-NI
H&E Staining : Intestinal Structure
treatment in Caco-2 cells after being damaged by 2% DSS. It implied that the
TNBS
No Treatment Diluent iCP-NI (IR) iCP-NI (IV) iCP-NI could recover the damaged internal layer of the intestines. This
hypothesis was validated via in vivo study of IBD mouse models, resulting in
reduced pathological scores in iCP-NI treatment groups. The expressions of
various pro-inflammatory cytokines were also significantly downregulated.
Scale bar = 200 μm
Wallace Score : Microscopic Score Ameho Score : Histological Score Overall, these results suggest that iCP-NI is a potential treatment for IBD.
P<0.001
***
* P<0.05
P<0.01 P<0.01 REFERENCES Contact Information
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TNBS TNBS