Plasma kallikrein (PKa) (IC50 = 6.0 nM in isolated enzyme assay; selectivity >6667-fold over tissue kallikrein, factor XIIa, and factor XIa)
Human plasma kallikrein (IC50 = 54 nM in human whole plasma activation assay) [1]

Sebetralstat demonstrated potent inhibitory activity against plasma kallikrein (PKa) with an IC50 of 6.0 nM in an isolated enzyme kinetic fluorogenic substrate assay. In a human whole plasma assay where the kallikrein-kinin system was activated by dextran sulfate, Sebetralstat inhibited PKa with an IC50 of 54 nM, representing a potency ratio of approximately 1:9 compared to the isolated enzyme IC50. Sebetralstat exhibited high selectivity (>40000 nM IC50) against a panel of related human serine proteases including tissue kallikrein (KLK1), factor XIIa, factor XIa, thrombin, trypsin, and plasmin. [1]
In vitro Caco-2 permeability assays showed Sebetralstat had an apparent permeability (Papp) of 9.0 × 10^-6 cm/s, indicating acceptable permeability. [1]
In vitro intrinsic clearance (CLint) in human liver microsomes (HLM) was measured at 14 µL/min/mg protein. [1]
Thermodynamic solubility assays in physiologically relevant media showed Sebetralstat had solubility above 0.1 mg/mL in both fasted state simulated intestinal fluid (FaSSIF) and fasted state simulated gastric fluid (FaSSGF). [1]

Pharmacokinetic studies in Sprague-Dawley rats following a single intravenous (1 mg/kg) or oral (10 mg/kg) dose showed a plasma clearance (CL) of 11.7 mL/min/kg, volume of distribution at steady state (Vss) of 0.50 L/kg, half-life (t1/2) of 1.0 h, and oral bioavailability (F) of 82%. [1]
Pharmacokinetic studies in beagle dogs following a single intravenous (1 mg/kg) or oral (1 mg/kg) dose showed a plasma clearance (CL) of 8.7 mL/min/kg, volume of distribution at steady state (Vss) of 0.65 L/kg, half-life (t1/2) of 1.0 h, and oral bioavailability (F) of 34%. [1]
Phase 1 clinical trials in healthy volunteers demonstrated rapid oral absorption of Sebetralstat and near-complete inhibition of plasma kallikrein enzyme activity in whole plasma. [1]

Plasma kallikrein (PKa) inhibitory activity was determined using a fluorogenic substrate cleavage assay. Human PKa was pre-incubated with test compounds for 5 minutes. Enzymatic activity was then analyzed kinetically by monitoring the cleavage of the fluorogenic substrate H-D-Pro-Phe-Arg-AFC using a fluorometer. The rate of fluorescence increase was measured. To estimate IC50 values, a four-parameter logistic dose-response curve was fitted to the normalized reaction rates. [1]
The inhibitory effects on a panel of related serine proteases (factor XIa, factor XIIa, tissue kallikrein-1, thrombin, trypsin, plasmin) were analyzed using similar fluorogenic substrate cleavage assays specific for each enzyme. Compounds were pre-incubated with each enzyme for 5 minutes prior to substrate addition, and IC50 values were determined by fitting dose-response curves. [1]
PKa activity in human whole plasma was measured using the same fluorogenic substrate (H-D-Pro-Phe-Arg-AFC). The kallikrein-kinin system in plasma was stimulated by adding dextran sulfate (DXS, 500 kDa) at a final concentration of 6.25 µg/mL. Compounds at various concentrations were pre-incubated in plasma for 5 minutes prior to DXS stimulation. PKa activity was estimated based on the maximum rate of fluorescence increase, and plasma IC50 values were determined. [1]

In vitro permeability was assessed using the Caco-2 cell monolayer model. Caco-2 cells were seeded on collagen-coated permeable filter inserts and maintained for differentiation. For the assay, test compounds were prepared in DMSO, diluted in buffer (pH 7.4), and added to the apical compartment. Buffer was placed in the basolateral compartment. Plates were incubated for 1 hour at 37°C with shaking. Samples were taken from both compartments after incubation, and the concentration of the test compound was quantified using LC-MS/MS. Apparent permeability (Papp) was calculated based on the transported amount, initial donor concentration, surface area, and incubation time. Monolayer integrity was verified by measuring transepithelial electrical resistance and Lucifer Yellow flux. [1]

Rat pharmacokinetic studies were performed in male Sprague-Dawley rats. For intravenous administration, a single dose of 1 mg/kg was administered as a 1 mg/mL formulation in vehicle at a volume of 1 mL/kg. For oral administration, a single dose of 10 mg/kg was administered as a 2 mg/mL formulation in vehicle at a volume of 5 mL/kg. Blood samples were collected at specified time points up to 24 hours post-dose for plasma concentration analysis. [1]
Dog pharmacokinetic studies were performed in male beagle dogs. For intravenous administration, a single dose of 1 mg/kg was administered as a 1 mg/mL formulation in vehicle at a volume of 1 mL/kg. For oral administration, a single dose of 1 mg/kg was administered as a 0.2 mg/mL formulation in vehicle at a volume of 5 mL/kg. Blood samples were collected at specified time points up to 24 hours post-dose for plasma concentration analysis. [1]

In Sprague-Dawley rats, the plasma clearance (CL) of sebetralstat was 11.7 mL/min/kg, approximately 20.9% of the rat liver blood flow (LBF, 80 mL/min/kg). The steady-state volume of distribution (Vss) was 0.50 L/kg. The terminal half-life (t1/2) was 1.0 h. The bioavailability (F) after oral administration of a 10 mg/kg dose was 82%. [1] In beagle dogs, the plasma clearance (CL) of sebetralstat was 8.7 mL/min/kg, approximately 33% of the canine liver blood flow (LBF, 33 mL/min/kg). The steady-state volume of distribution (Vss) was 0.65 L/kg. The terminal half-life (t1/2) was 1.0 h. The oral bioavailability (F) was 34% (1 mg/kg dose). [1]
In vitro experiments showed that sebetralstat had a moderate intrinsic clearance rate in human liver microsomes (HLM CLint = 14 µL/min/mg protein). [1]
Sebetralstat had good permeability in Caco-2 cells (Papp = 9.0 × 10^-6 cm/s). [1]
Sebetralstat had good thermodynamic solubility in physiologically relevant media, with solubility exceeding 0.1 mg/mL in both FaSSIF and FaSSGF. [1]

The inhibitory effect of sebetralstat on hERG potassium channels was tested using whole-cell patch-clamp technique. The results showed that the IC50 was > 33 µM, indicating a low risk of QT interval prolongation. [1]
Inhibition assays against seven major human cytochrome P450 (CYP450) isoenzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4) showed that the IC50 values of all isoenzymes were > 25 µM, indicating a very low risk of CYP-mediated drug interactions. [1]
Ames assays on non-GLP bacterial mutagenicity showed that sebetralstat had no risk of genotoxicity. [1]

[1]. Sebetralstat (KVD900): A Potent and Selective Small Molecule Plasma Kallikrein Inhibitor Featuring a Novel P1 Group as a Potential Oral On-Demand Treatment for Hereditary Angioedema. J Med Chem. 2022 Oct 27;65(20):13629-13644.

[2]. WO2016083820.

Sebetricostat (KVD900) is a potent, selective, and orally bioavailable small molecule inhibitor of kallikrein (PKa). It was designed for on-demand treatment of acute exacerbations of hereditary angioedema (HAE). The drug development process involved systematically reducing the basicity of the P1 group to improve its oral drug properties, ultimately resulting in a novel 3-fluoro-4-methoxypyridyl P1 group. When secbetricostat binds to PKa, it forms a characteristic U-shaped conformation due to the inversion of the Trp215 rotomer, resulting in π-π stacking interactions and a hydrogen bond network, but lacking conventional ionic interactions with Asp189 in the S1 pocket. Its binding affinity is attributed to hydrophobic interactions and the substitution of high-energy water molecules near Tyr228. Based on encouraging preclinical data, secbetricostat has entered clinical trials. Phase I trials in healthy volunteers have demonstrated its rapid absorption and near-complete PKa inhibition. The drug has also been evaluated in Phase II and Phase III clinical studies for hereditary angioedema (HAE). [1] Sebetrolstat is a plasma kallikrein inhibitor. Sebetrolstat works by acting as a kallikrein inhibitor. Sebetrolstat is a small molecule drug with Phase III clinical trials covering all indications and two investigational indications. The efficacy and safety of EKTERLY have been demonstrated by the results of the Phase III KONFIDENT clinical trial conducted by KalVista, which is the largest clinical trial program in the field of HAE to date. In May 2024, the New England Journal of Medicine published data from the KONFIDENT study, which showed that compared with placebo, EKTERLY significantly relieved symptoms faster, reduced the severity of attacks and accelerated the resolution of attacks, and was well tolerated with a safety profile similar to placebo. ² This trial randomized 136 patients with hereditary angioedema (HAE) from 66 clinical centers in 20 countries. The more real-world-oriented KONFIDENT-S open-label expansion trial further supports these results. As of September 2024, this trial showed that EKTERLY enabled patients to initiate treatment within an average of 10 minutes after an attack. Recent data from KONFIDENT-S showed a median time to symptom relief of 1.3 hours for breakthrough attacks involving the larynx, abdomen, and in patients receiving long-term prophylactic treatment. In the KONFIDENT-S study, the safety profile of EKTERLY 600 mg was consistent with that observed in the KONFIDENT study, which included a larger number of attack events (>1700).

C26H26FN5O4

491.514149188995

491.196

C, 62.89; H, 5.07; F, 3.98; N, 14.67; O, 13.40

1933514-13-6

2163789-76-0 (HCl); 2163789-80-6; 1933514-13-6; 2163789-82-8 (besylate); 2163789-79-3 (mesylate)

121365142

White to off-white solid powder

1.3

1

7

10

36

803

0

FC1=C(C=CN=C1CNC(C1=CN(CC2C=CC(=CC=2)CN2C=CC=CC2=O)N=C1COC)=O)OC

KGMPDQIYDKKXRD-UHFFFAOYSA-N

InChI=1S/C26H26FN5O4/c1-35-17-22-20(26(34)29-13-21-25(27)23(36-2)10-11-28-21)16-32(30-22)15-19-8-6-18(7-9-19)14-31-12-4-3-5-24(31)33/h3-12,16H,13-15,17H2,1-2H3,(H,29,34)

N-[(3-fluoro-4-methoxypyridin-2-yl)methyl]-3-(methoxymethyl)-1-[[4-[(2-oxopyridin-1-yl)methyl]phenyl]methyl]pyrazole-4-carboxamide

Sebetralstat; 1933514-13-6; KVD900; KVD-900; O5ZD2TU2B7;

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)

DMSO: ≥ 125 mg/mL (254.32 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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