Endothelial lipase (EL) [1]
EL: IC50 = 0.237 μM (human, cell-free FELA mixed micelle assay) [1]
EL: IC50 = 0.11 μM (human, functional HDL particle assay) [1]
EL: IC50 = 0.25 μM (human, cell-based assay using EL-transfected HEK cells and PED-A substrate) [1]
LPL: IC50 = 209.5 μM (human) [1]
XEN445 targets endothelial lipase, an enzyme that hydrolyzes phospholipids and is a key regulator of HDL metabolism. Endothelial lipase is a member of the triglyceride lipase gene family and is primarily expressed in the vascular endothelium. By inhibiting this enzyme, XEN445 prevents the hydrolysis of HDL phospholipids, leading to an increase in HDL particle size and concentration. This mechanism has made it a target for research into raising HDL cholesterol levels for the treatment of cardiovascular disease.

XEN445 shows good in vitro inhibitory potency against EL, and high selectivity over LPL and HL.
Cell Assay: In vitro, XEN445 has been reported to selectively inhibit EL with IC50 values of 0.237μM, 20μM and 9.5μM for hEL, hLPL and hHL, respectively. In addition, XEN445 has been revealed to suppress EL in EL-transfected HEK cells with an IC50 value of 0.25μM which is very similar to that tested in cell-free assay. Besides, XEN445 has shown significantly high oral bioavailability and low clearance rates in C57BL6 mice.

---

XEN445 exhibited potent inhibitory activity against human endothelial lipase (EL) in a cell-free fluorogenic phosphatidylcholine mixed micelle assay (FELA, bis-BODIPY-FL C11-PC), with an IC50 of 0.237 μM [1].
In a more physiological functional HDL particle assay using purified human HDL as substrate and measuring free fatty acid release, XEN445 showed an IC50 of 0.11 μM [1].
In a cell-based assay using EL-transfected HEK cells and the soluble EL fluorogenic substrate PED-A, after 30 min preincubation of cells with XEN445, an IC50 value of 0.25 μM was obtained, similar to the cell-free assay [1].
Selectivity: XEN445 demonstrated high selectivity over human lipoprotein lipase (LPL, IC50 = 209.5 μM) with >880-fold selectivity; selectivity over human hepatic lipase (HL) was not determined but compounds in this class generally showed good selectivity [1].
In vitro, XEN445 has been shown to be a potent inhibitor of endothelial lipase with an IC50 of 0.237 µM. It is selective for endothelial lipase over other lipases, such as hepatic lipase and lipoprotein lipase. The compound's activity has been characterized in enzyme assays using purified or recombinant endothelial lipase. It is a valuable tool for studying the function of endothelial lipase in vitro.

In vivo, XEN445 displays good ADME and PK properties. In mice, XEN445 (30 mg/kg, p.o.) raises plasma HDL-cholesterol concentration.
Oral administration of XEN445 at 30 mg/kg twice daily (b.i.d.) for 3 days to wild-type C57BL/6 mice raised total plasma cholesterol by 18% and plasma HDL-cholesterol (HDLc) by 16% compared to vehicle-treated wild-type mice, with average plasma levels of XEN445 at termination (16 h post final dose) of 9.9 μM [1].
Dosing XEN445 at 10 mg/kg b.i.d. for 3 days to wild-type mice resulted in approximately 10% increase in plasma HDLc [1].
To confirm on-target efficacy, XEN445 was dosed to EL knockout (LIPG KO) mice at 30 mg/kg b.i.d. for 3 days; no significant change in total plasma cholesterol or HDLc was observed compared to vehicle-treated EL knockout mice, while vehicle-treated EL knockout mice had 121% higher HDLc than wild-type controls [1].
Extended treatment of wild-type mice with XEN445 at 30 mg/kg b.i.d. for 9 days resulted in a 30% increase in plasma HDLc (vs. 16% at 3 days) [1].
In vivo, XEN445 is an orally active compound that has been shown to increase HDL cholesterol levels in animal models. Studies in mice have demonstrated that administration of XEN445 leads to a significant increase in plasma HDL cholesterol. This effect is attributed to the inhibition of endothelial lipase in the vascular endothelium. The compound has been investigated for its potential to treat dyslipidemia and reduce the risk of cardiovascular disease.

The activity of endothelial lipase (EL) was measured using a mixed micelle fluorogenic phosphatidylcholine/Triton X-100 assay. Recombinant human or mouse EL was diluted in PBS containing 14% DMEM, 2% glycerol. 40 μL of diluted enzyme (1.4 nM final concentration) was added to each well of a 96-well plate containing inhibitors or vehicle (5 μL in 10% DMSO). The enzyme-inhibitor mixture was incubated at room temperature for 30 min, then 5 μL of 10 μM bis-BODIPY-FL C11-PC in 0.1% Triton X-100 was added. Fluorescence was read at excitation 490 nm and emission 520 nm. Human LPL (4 nM final) assay required addition of ApoCII (1 μg/μL final). Human HL (6 nM final) was assayed in 50 mM Tris pH 8.5, 1 M NaCl. Background activity was determined using mock-transfected HEK-293 cell media [1].
Functional HDL particle assay: Potency of EL inhibitors was determined using purified human HDL particles as a physiologically relevant substrate. Release of free fatty acids was measured as readout of lipase activity [1].
Cell-based EL activity assay: EL-transfected HEK cells were preincubated with XEN445 for 30 min, then the soluble EL fluorogenic substrate PED-A was added. The IC50 was determined [1].
Recombinant human and mouse EL, human LPL, and human HL were obtained by transient transfection of HEK-293 cells with codon-optimized sequences in pcDNA3.1. Cells were grown in DMEM +10% FBS to 50-80% confluency in 225 cm² flasks and transfected with 95 μg plasmid DNA using Lipofectamine. After 5 h, media was replaced with Opti-MEM and cells cultured for 72 h. Cell-associated lipase was displaced by adding heparin to 50 U/mL for 30 min. Media was removed and concentrated 100-fold using concentrators (30 kDa cutoff). Active site concentration of each lipase was determined using orlistat titration [1].
Non-cellular enzyme assays for XEN445 involve measuring its inhibition of endothelial lipase activity. These assays typically use purified or recombinant endothelial lipase and a synthetic substrate, such as a fluorescently labeled phospholipid. The rate of substrate hydrolysis is measured in the presence of varying concentrations of the compound to determine the IC50. These experiments are essential for characterizing its potency and selectivity.

XEN445 was tested in a cell-based assay using EL-transfected HEK cells. After 30 min preincubation of EL-expressing HEK cells with XEN445, the soluble EL fluorogenic substrate PED-A was added, and an IC50 value of 0.25 μM was obtained, similar to that determined in the cell-free assay [1].
In vitro cell-based assays for XEN445 are used to study its effects on endothelial cells. These assays often involve culturing endothelial cells and measuring the activity of endothelial lipase in the presence of the compound. The effects on HDL metabolism and lipid efflux can also be studied in cell-based systems. These experiments help to confirm its mechanism of action at the cellular level.

Formulated in 0.2% Tween-20/1% carboxymethyl-cellulose; 30 mg/kg bid; p.o. Male EL knockout mice and age-matched wild-type male C57BL6 mice

---

Mouse pharmacokinetics: Male C57BL/6 mice (non-fasted, n=3) were administered XEN445 orally (po) at 10 mg/kg in 50% PEG 400 and 50% D5W (dextrose 5% in water), or intravenously (iv) at 1 mg/kg in 10% ethanol, 25% PEG 400 and 65% D5W. Pharmacokinetic parameters were determined [1].
In vivo efficacy study: Male C57BL/6 wild-type or EL knockout (LIPG KO) mice were orally dosed with XEN445 at 30 mg/kg or 10 mg/kg twice daily (b.i.d.) for 3 or 9 days. Vehicle groups received the same dosing regimen without compound. Blood was collected on the morning of day 4 (or day 10) at 16 h post final dose. Plasma total cholesterol and HDLc concentrations were measured [1].
In vivo animal studies for XEN445 have been conducted in mouse models. The compound is typically administered orally to evaluate its effects on plasma HDL cholesterol levels. Blood samples are collected at various time points to measure lipid profiles. These studies are crucial for validating its in vivo efficacy as a potential therapeutic agent for dyslipidemia.

Mouse pharmacokinetic parameters of XEN445 (iv 1 mg/kg, po 10 mg/kg in male C57BL/6 mice, n=3): Cmax (po) = 6.2 μM; Tmax (po) = 0.5 h; Cmin at 24 h (po) = 4.5 μM; AUC last (po) = 47 μM·h; AUC last (iv) = 25.3 μM·h; terminal half-life (t1/2, po) = 11.9 h; clearance (CL, iv) = 0.06 L/h/kg; oral bioavailability (F) = 54% [1].
XEN445 is an orally available compound with a molecular weight of 418.41 g/mol. As a research compound, its pharmacokinetic properties have been studied in preclinical models. It is expected to be absorbed after oral administration and to be distributed to tissues, particularly the vascular endothelium where its target is located. Its half-life and bioavailability would be key parameters for its development.

Plasma protein binding (PPB): XEN445 showed high affinity for rat plasma proteins (97.4% bound at 2.5 μM compound in 10% rat plasma/90% PBS by equilibrium dialysis for 4 h at 37°C), mouse plasma proteins (88.8% bound), and human plasma proteins (95.4% bound) [1].
Comprehensive toxicological data for XEN445 are not extensively detailed in standard summaries, as it is a research compound. As with all investigational drugs, its safety profile would need to be evaluated in preclinical studies. XEN445 is not an approved drug and is available only as a research compound.

Bioorg Med Chem.2013 Dec 15;21(24):7724-34.

XEN445 (compound 13) is a potent and selective endothelial lipase (EL) inhibitor belonging to the anthranilic acid class. It was discovered through optimization of a high-throughput screening hit (compound 1) with focus on safety, potency, selectivity, and protein binding. EL hydrolyzes phosphatidylcholine in HDL particles, reducing HDL particle size. EL activity is inversely correlated with plasma HDLc levels. XEN445 demonstrated in vivo efficacy in raising plasma HDLc in wild-type mice, and the effect was confirmed to be on-target using EL knockout mice. This compound is useful for further defining catalytic and non-catalytic roles of EL in HDL metabolism and as a starting point for developing EL-directed therapeutics for dyslipidemia-related cardiovascular disease [1].
XEN445 is a potent, selective, and orally available inhibitor of endothelial lipase with an IC50 of 0.237 µM. It is used in research to study the role of endothelial lipase in HDL metabolism. By inhibiting this enzyme, it increases HDL cholesterol levels in animal models. XEN445 has been investigated for its potential to treat dyslipidemia. It is not an approved drug and is available only as a research compound.

C18H17F3N2O3

366.33

366.119

C, 59.02; H, 4.68; F, 15.56; N, 7.65; O, 13.10

1515856-92-4

72736173

White to off-white solid powder

1.4±0.1 g/cm3

490.9±45.0 °C at 760 mmHg

250.7±28.7 °C

0.0±1.3 mmHg at 25°C

1.583

3.3

1

8

5

26

489

1

FC(C1C([H])=C([H])C(=C(C(=O)O[H])C=1[H])N1C([H])([H])C([H])([H])[C@@]([H])(C1([H])[H])OC([H])([H])C1=C([H])C([H])=C([H])C([H])=N1)(F)F

NBGRERFNOKZQLO-AWEZNQCLSA-N

InChI=1S/C18H17F3N2O3/c19-18(20,21)12-4-5-16(15(9-12)17(24)25)23-8-6-14(10-23)26-11-13-3-1-2-7-22-13/h1-5,7,9,14H,6,8,10-11H2,(H,24,25)/t14-/m0/s1

(S)-2-(3-(pyridin-2-ylmethoxy)pyrrolidin-1-yl)-5-(trifluoromethyl)benzoic 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)

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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