UK-371804: Selective inhibitor of urokinase-type plasminogen activator (uPA) with a Ki value of 10 nM; it exhibits high selectivity, showing 4000-fold selectivity over tissue-type plasminogen activator (tPA) and 2700-fold selectivity over plasmin [1]

In human chronic wound fluid, UK-371804 can inhibit exogenous uPA (IC50=0.89 μM). Outstanding enzyme potency (Ki=10 nM) and selectivity profile (4000-fold versus tPA and 2700-fold versus plasmin) are characteristics of UK-371804[1].

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Enzyme inhibitory activity: UK-371804 is a potent and highly selective inhibitor of urokinase-type plasminogen activator (uPA). In the enzyme activity assay, it has a Ki value of 10 nM, demonstrating strong binding affinity to uPA. It shows excellent selectivity, with 4000-fold higher inhibitory activity against uPA compared to tPA and 2700-fold higher than plasmin [1]
- Inhibition of uPA in human chronic wound fluid: In an in vitro assay using human chronic wound fluid, UK-371804 effectively inhibits exogenous uPA activity present in the fluid, with an IC50 value of 0.89 μM [1]

After topical application, UK-371804 can penetrate pig wounds in a porcine acute excisional wound model and decrease exogenous uPA activity without negatively affecting wound healing parameters. In the dermis, UK-371804 concentrations are 41.8 μM[1].

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Activity in porcine acute excisional wound model: In a porcine acute excisional wound model, UK-371804 is administered topically. The drug can penetrate into the pig wounds and effectively inhibit exogenous uPA activity. Importantly, topical application of UK-371804 does not have any adverse effects on wound healing parameters, such as wound closure rate or tissue regeneration [1]

uPA, tPA, and plasmin inhibitory activity assay:
1. Preparation of enzymes and substrates: Purify urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasmin to obtain active enzyme preparations. Prepare specific chromogenic or fluorogenic substrates for each enzyme, which can release a detectable signal upon cleavage by the corresponding enzyme.
2. Reaction setup: For each enzyme (uPA, tPA, plasmin), prepare reaction mixtures containing the enzyme, appropriate buffer, and different concentrations of UK-371804. Incubate the mixtures at a suitable temperature (e.g., 37°C) for a certain period to allow the inhibitor to interact with the enzyme.
3. Substrate addition and detection: Add the specific substrate for each enzyme to the corresponding reaction mixture and continue incubation. Monitor the signal generated by substrate cleavage (e.g., absorbance or fluorescence intensity) over time using a microplate reader to measure enzyme activity.
4. Data analysis: Calculate the enzyme activity in the presence of different concentrations of UK-371804 relative to the control group (without inhibitor). Use kinetic analysis methods to determine the Ki value of UK-371804 for uPA (10 nM) and calculate the selectivity ratios relative to tPA (4000-fold) and plasmin (2700-fold) [1]
- uPA inhibition assay in human chronic wound fluid:
1. Sample preparation: Collect human chronic wound fluid and centrifuge or filter to remove debris and impurities, obtaining a clear fluid sample.
2. Reaction setup: Prepare reaction mixtures containing the chronic wound fluid (which contains exogenous uPA), reaction buffer, and different concentrations of UK-371804. Set up a control group without the inhibitor.
3. Substrate addition and incubation: Add a uPA-specific substrate to the reaction mixtures and incubate at an appropriate temperature for a specified time to allow the reaction to proceed.
4. Detection and IC50 calculation: Measure the signal from substrate cleavage to assess the remaining uPA activity in each reaction mixture. Plot the dose-response curve based on the inhibitor concentrations and corresponding uPA activity, and calculate the IC50 value (0.89 μM) of UK-371804 for inhibiting uPA in human chronic wound fluid [1]

Two female pigs are subjected to eight excisional wounds. The wounds are dressed and treated daily for 10 days with either 1 mL of a 10 mg/mL formulation of UK-371804 in hydrogel vehicle.
Pig

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Porcine acute excisional wound model experiment:
1. Animal preparation: Use pigs as experimental animals and acclimate them to the laboratory environment for a period before the experiment to ensure their physical condition is stable.
2. Wound creation: Create acute excisional wounds of uniform size and depth on the designated area of the pigs' skin using a standardized surgical method. Ensure the wounds are consistent in terms of location, size, and depth across all experimental animals.
3. Drug preparation and administration: Prepare UK-371804 into a suitable topical formulation (e.g., cream, gel, or solution) at the desired concentration. Apply the drug formulation topically to the created wounds according to a predetermined schedule (e.g., once daily for a specific number of days). Set up a control group that receives a vehicle formulation without the drug.
4. Sample collection and detection: At specified time points after drug administration, collect wound tissue samples or wound fluid (if applicable) to detect uPA activity. Use appropriate biochemical assays to measure the inhibitory effect of UK-371804 on exogenous uPA activity in the wounds.
5. Wound healing assessment: Regularly observe and record wound healing parameters, such as wound closure rate, epithelialization, and tissue granulation formation. At the end of the experiment, perform histological analysis of the wound tissue to evaluate the effect of the drug on wound healing at the tissue level [1]

In vivo safety assessment in a porcine wound model: Topical application of UK-371804 in a porcine acute excision wound model did not have any adverse effects on wound healing parameters. No evidence of delayed wound closure, abnormal tissue regeneration, or local toxicity at the wound site (such as inflammation, redness, or swelling) was found [1].

[1]. Selective urokinase-type plasminogen activator inhibitors. 4. 1-(7-sulfonamidoisoquinolinyl)guanidines. J Med Chem. 2007 May 17;50(10):2341-51.

Chemical structure and optimization: UK-371804 belongs to the 1-(7-sulfonamidoisoquinolinyl)guanidine series of compounds. It was developed by introducing a 7-sulfonamide group to improve efficacy and selectivity using 1-isoquinolinylguanidine as a template. The sulfonamide moiety derived from amino acids further improves its pharmacological properties [1]
- Binding mode study: Through X-ray co-crystallization studies, we studied the binding mode of UK-371804 with uPA, thereby gaining a deeper understanding of its high selectivity and efficacy for uPA, laying the foundation for further optimization of the structure of uPA inhibitors [1]
- Therapeutic potential: Based on its strong uPA inhibitory activity, high selectivity, ability to penetrate wound tissue and safety in animal models, UK-371804 has been selected as a candidate drug for further preclinical evaluation and is expected to be used to treat chronic skin ulcers [1]

C14H16CLN5O4S

385.82

385.06

C, 43.58; H, 4.18; Cl, 9.19; N, 18.15; O, 16.59; S, 8.31

256477-09-5

256477-09-5;256476-36-5 (HCl);

9952109

White to off-white solid powder

0.7

4

7

5

25

644

0

XSDAXWRCPTYNOD-UHFFFAOYSA-N

InChI=1S/C14H16ClN5O4S/c1-14(2,12(21)22)20-25(23,24)7-3-4-8-9(5-7)11(19-13(16)17)18-6-10(8)15/h3-6,20H,1-2H3,(H,21,22)(H4,16,17,18,19)

2-[[4-chloro-1-(diaminomethylideneamino)isoquinolin-7-yl]sulfonylamino]-2-methylpropanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: 0.83 mg/mL (2.15 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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.

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Solubility in Formulation 2: ≥ 0.83 mg/mL (2.15 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 8.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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