Protein arginine deiminase 4 (PAD4); IC50 = 5.9 ± 0.3 μM [1]

Pan-PAD inhibitor (inhibits all active PAD isozymes with near equal potency); kinact/KI = 13,000 M⁻¹·min⁻¹ for PAD4 [1]

Also inhibits PAD2 and PAD4 expressed in inflammatory cells [2]

Cl-amidine hydrochloride is a haloacetamidine-based bioavailable drug that efficiently inhibits all active PAD isoenzymes (kinact/KI=13,000 M-1 min-1 for PAD4) [1 Cl-amidine hydrochloride (0, 5, 10, 15, 20, 25, 50 μg/mL, 24 hours) induces HT29 and TK6 lymphoblastoid cells. It displays the dose diagram on the round cell mold. compared to cells generated by Cl-amidine hydrochloride, the duct amidine series (HT29) is comparatively resistant [2]. In animal models, clonidine hydrochloride improves and inhibits neutrophil extracellular tray creation and histone 3 acidification [4].

---

Cl-amidine irreversibly inactivates PAD4 in a time- and concentration-dependent manner. The inactivation is calcium-dependent, with >10-fold preference for the calcium-bound form of the enzyme. Substrate (BAEE) protects against inactivation, consistent with active site modification. Rapid dilution and dialysis experiments show no recovery of enzymatic activity, confirming irreversible inactivation. Kinetic parameters: kinact = 2.4 ± 0.2 min⁻¹, KI = 180 ± 33 μM, kinact/KI = 13,000 M⁻¹·min⁻¹ [1]

Cl-amidine antagonizes the PAD4-mediated enhancement of the p300GBD-GRIP1 interaction in CV-1 cells in a dose-dependent manner (0–200 μM) [1]

Cl-amidine induces apoptosis in TK6 lymphoblastoid cells in a dose-dependent manner as assessed by annexin V/PI staining (0–50 μg/ml over 24 h). In contrast, HT29 colon cancer cells (p53 mutant) are relatively resistant to Cl-amidine-induced apoptosis [2]

In CD45⁺ inflammatory cells isolated from mouse colons, Cl-amidine treatment (75 mg/kg ip or po) elevates p53 levels, increases p21WAF1 expression, and induces PARP cleavage, indicating cell cycle arrest and apoptosis [2]

Cl-amidine (75 mg/kg, intraperitoneal injection once daily) prevents and cures mice's DSS-induced cystitis [2]. Histological scores significantly decreased in response to clonidine (5, 25, 75 mg/kg, once daily via side wall gavage) in a dose-dependent manner [2].

---

In a mouse DSS-induced colitis model, prophylactic intraperitoneal administration of Cl-amidine (75 mg/kg/day, starting at day 0) significantly reduces colon inflammation (histology score 13.9±1.6 vs 24.2±1.7 for DSS alone), prevents DSS-induced colon shortening (colon length 8.4±0.2 cm vs 7.2±0.2 cm for DSS alone), and improves overall health (total distance traveled 3,190±401 m vs 800±163 m for DSS alone) [2]

Therapeutic oral gavage of Cl-amidine (5, 25, 75 mg/kg/day) starting at day 7 after colitis onset reduces histology scores in a dose-dependent manner (17.4±3.7, 15.5±2.3, and 3.1±0.3, respectively, compared to 34.8±1.4 for DSS alone at 2 weeks). Cl-amidine treatment prevents weight loss and colon shrinkage and does not significantly alter white blood cell counts compared to water-treated controls [2]

Cl-amidine treatment (75 mg/kg ip) significantly reduces PAD activity and total protein citrullination in colons of DSS-treated mice. Peptidylcitrulline content is also decreased. PAD levels (pan-PAD immunoreactivity score) are elevated in DSS colitis and suppressed by Cl-amidine [2]

Cl-amidine increases apoptosis of mesenteric lymph node (MLN) inflammatory cells in DSS-induced colitis as measured by TUNEL assay (immunoreactivity score significantly elevated in DSS+Cl-amidine group compared to DSS alone). In contrast, epithelial cell apoptosis is reduced in Cl-amidine-treated mice, indicating protection of normal colon epithelium [2]

IC50 determination: PAD4 was preincubated with various concentrations of Cl-amidine in the presence of 10 mM calcium for 15 min at 37°C. The reaction was initiated by adding 10 mM BAEE. After incubation, the amount of citrulline produced was quantified. Concentration-response data were fitted to the equation fractional activity = 1/(1+[I]/IC50) [1]

Time course inactivation: PAD4 was incubated with different concentrations of Cl-amidine in assay buffer containing BAEE. At various time points, aliquots were withdrawn and enzyme activity quenched by flash-freezing. Citrulline production was quantified. Progress curves were fitted to [Cit] = v₁(1-e^{-k_obs t})/k_obs to obtain pseudo-first-order rate constants. Values of k_obs were plotted against Cl-amidine concentration and fitted to k_obs = k_inact[I]/(K_I+[I]) [1]

Rapid dilution assay: Preformed PAD4-Cl-amidine complexes (generated by incubating enzyme with 250 μM Cl-amidine for 30 min at 37°C) were diluted 100-fold into assay buffer containing 10 mM BAEE. Product formation was monitored over time. No recovery of activity was observed, indicating irreversible inactivation [1]

Dialysis experiment: Preformed PAD4-Cl-amidine complexes were dialyzed against buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 500 mM NaCl, 1 mM DTT, 10% glycerol) for 3.5 h and then for an additional 16.5 h. No recovery of enzymatic activity was observed at either time point, ruling out slow-binding reversible inhibition [1]

Calcium dependence: PAD4 was preincubated with Cl-amidine in the absence or presence of calcium before adding BAEE. Calcium was added to the calcium-free preincubation sample at the start of the assay to activate PAD4. Cl-amidine preferentially inactivated the calcium-bound form of the enzyme by >10-fold [1]

PAD activity measurement in tissue samples: Colon or spleen lysates were added to reaction buffer containing 50 mM NaCl, 10 mM CaCl₂, 2 mM DTT, 100 mM Tris pH 7.6, and 10 mM BAEE. Reactions proceeded for 2 h at 37°C, then frozen. COLDER color development solution (detects citrullinated proteins) was added, incubated at 95°C for 30 min, and absorbance at 540 nm measured. Background was subtracted using sample blanks, and data normalized to protein concentration [2]

Peptidylcitrulline measurement: Tissue lysates were desalted using Sephadex G-10 columns to remove free citrulline. Desalted samples were reacted with COLDER solution, incubated at 95°C for 30 min, and absorbance measured at 540 nm. Background subtracted using resuspension buffer and normalized to protein concentration [2]

Apoptosis analysis[2].
Cell Types: TK6 lymphoblastoid cells and HT29 colon cancer cells.
Tested Concentrations: 0, 5, 10, 15, 20, 25, 50 μg/mL.
Incubation Duration: 24 hrs (hours).
Experimental Results: The induction of apoptosis was dose-dependent.

---

Mammalian two-hybrid assay: CV-1 cells were transiently transfected with plasmids encoding a luciferase reporter construct, p300GBD fused to the Gal4 DNA binding domain, the GRIP1 AD1 domain fused to the VP16 activation domain, and either wild-type PAD4 or catalytically defective C645S mutant. Transfections proceeded for 3 h, then medium was replaced. Cl-amidine dissolved in 10 mM Na-HEPES (pH 7.0) was added at concentrations ranging from 0 to 200 μM. After 40 h incubation, cell extracts were prepared and luciferase activity quantified. Cl-amidine antagonized PAD4-mediated enhancement of p300GBD-GRIP1 interaction in a dose-dependent manner, with minimal reduction in C645S-transfected cells [1]

Apoptosis assay in TK6 and HT29 cells: Cells were seeded at 1×10⁶ cells/well for 24 h. Concanavalin A (2.5 μg/ml) was added for 12 h to activate TK6 cells, then washed off. Fresh medium containing Cl-amidine (0–50 μg/ml) was added for 24 h. Cells were harvested and stained with annexin V/propidium iodide, then analyzed by flow cytometry. TK6 cells showed dose-dependent apoptosis, while HT29 cells were relatively resistant [2]

TUNEL assay on tissue sections: Serial colon sections from treated mice were subjected to terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Biotinylated nucleotide was incorporated at 3’-OH DNA ends, then streptavidin-HRP bound and detected with diaminobenzidine. Apoptotic nuclei stained dark brown. Counterstain was CAT Hematoxylin. TUNEL was quantified in epithelial areas and mesenteric lymph nodes. Cl-amidine increased apoptosis of MLN inflammatory cells but reduced epithelial cell apoptosis compared to DSS alone [2]

Western blot analysis of CD45⁺ inflammatory cells: Colonic mucosal cells were isolated, and CD45⁺ inflammatory cells were purified using magnetic microbead positive selection. Cell lysates were probed with antibodies against p53, p21WAF1/Cip1, and PARP. Cl-amidine treatment (75 mg/kg ip or po) elevated p53 levels, increased p21, and induced PARP cleavage, indicating cell cycle arrest and apoptosis [2]

Animal/Disease Models: C57BL/6 mice (8-12 weeks old, DSS colitis mouse model) [2].
Doses: 75 mg/kg. Management: IP one time/day.
Experimental Results: Inhibition of PAD activity in vivo, protein citrullination, and PAD levels in the colon.

---

Animal/Disease Models: C57BL/6 mice (8-12 weeks old, DSS colitis mouse model) [2].
Doses: 5, 25, 75 mg/kg.
Route of Administration: po (oral gavage), one time/day.
Experimental Results: Resultant in Dramatically lower histological scores.

---

For prophylactic treatment in DSS-induced colitis model: C57BL/6 mice (8–12 weeks old) received 2% dextran sulfate sodium (DSS) in drinking water from day 0. Cl-amidine was dissolved in 1× PBS and administered by intraperitoneal injection daily at 75 mg/kg body weight (human equivalent dose 6.1 mg/kg daily) starting at day 0. On day 14, blood, spleen, and colon samples were collected. Colon length was measured, and colons were processed for histology (H&E staining), immunohistochemistry, and inflammatory cell isolation. Histology score was calculated by multiplying percent involvement for inflammation severity (0–3), extent (0–3), and crypt damage (0–4) by percent area involvement (0–4) [2]

For therapeutic treatment (oral gavage) after colitis onset: Mice received 2% DSS for 1 week to induce moderate colitis. Starting at day 7, Cl-amidine (dissolved in 1× PBS) was administered by oral gavage daily at doses of 5, 25, or 75 mg/kg while continuing DSS for another week. On day 14, colons were collected and analyzed. Cl-amidine reduced histology scores in a dose-dependent manner, prevented weight loss and colon shrinkage, and did not significantly alter white blood cell counts compared to water-treated controls [2]

No animal deaths occurred during the study regardless of treatment. No gross abnormalities were observed in tissues from Cl-amidine-treated groups (water+Cl-amidine and DSS+Cl-amidine). The water+Cl-amidine group gained more weight (4.1±0.7 g) than the water-only group (2.8±0.4 g). Total white blood cell counts did not differ significantly between water-treated (5,100±600 WBCs/μl) and water+Cl-amidine (5,900±400 WBCs/μl) groups [2]

No generalized immunosuppressive effects were observed with Cl-amidine treatment. No signs of toxicity were noted even when given daily for 56 days at doses as high as 100 mg/kg/day in a previous RA model [2]

[1]. Inhibitors and Inactivators of Protein Arginine Deiminase 4: Functional and Structural Characterization. Biochemistry. 2006 Oct 3; 45(39): 11727–11736.

[2]. Suppression of colitis in mice by Cl-amidine: a novel peptidylarginine deiminase inhibitor. Am J Physiol Gastrointest Liver Physiol. 2011 Jun;300(6):G929-38.

[3]. Molecular targeting of protein arginine deiminases to suppress colitis and prevent colon cancer. Oncotarget. 2015 Nov 3;6(34):36053-62.

[4]. Cl-Amidine Prevents Histone 3 Citrullination and Neutrophil Extracellular Trap Formation, and Improves Survival in a Murine Sepsis Model. J Innate Immun. 2017;9(1):22-32.

Cl-amidine is a haloacetamidine-based irreversible inactivator of PAD4. It specifically modifies the active site cysteine (Cys645) via covalent bond formation. The irreversible nature is due to the formation of a stable thioether adduct. Cl-amidine is bioavailable and shows enhanced potency compared to F-amidine (IC50 5.9 μM vs 21.6 μM for F-amidine) [1]

Dysregulated PAD4 activity is implicated in rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Cl-amidine suppresses colitis in the DSS mouse model and reduces disease severity in the murine collagen-induced arthritis model of RA. These findings validate PADs as therapeutic targets for IBD and RA [1][2]

PAD levels (PAD2 and PAD4) are elevated in mouse and human colitis tissues. Cl-amidine, as a pan-PAD inhibitor, reduces PAD activity, protein citrullination, and PAD levels in vivo. The compound induces apoptosis of inflammatory cells (e.g., in mesenteric lymph nodes) via a p53-dependent pathway (elevation of p53, p21, and PARP cleavage), providing a mechanism for its anti-inflammatory effects [2]

C14H20CL2N4O2

347.24

346.096

1373232-26-8

D-Cl-amidine hydrochloride;Cl-amidine;913723-61-2;Cl-amidine TFA;1043444-18-3;D-Cl-amidine;1404060-15-6

74889978

White to light yellow solid powder

4

3

8

22

381

1

C1=CC=C(C=C1)C(=O)N[C@@H](CCCN=C(CCl)N)C(=O)N.Cl

OPFMEGSAOZAJIV-MERQFXBCSA-N

InChI=1S/C14H19ClN4O2.ClH/c15-9-12(16)18-8-4-7-11(13(17)20)19-14(21)10-5-2-1-3-6-10;/h1-3,5-6,11H,4,7-9H2,(H2,16,18)(H2,17,20)(H,19,21);1H/t11-;/m0./s1

N-[(1S)-1-(Aminocarbonyl)-4-[(2-chloro-1-iminoethyl)amino]butyl]-benzamide Hydrochloride

Cl amidine hydrochlorideCl amidine HClCl-amidine HCl Cl-amidine hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~143.99 mM)
H2O : ~50 mg/mL (~143.99 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (3.60 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 1.25 mg/mL (3.60 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 1.25 mg/mL (3.60 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 5.5 mg/mL (15.84 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)