5-HT2B ( IC50 = 3.4 )
PRX-08066 Maleic acid is a potent and selective antagonist of the human 5-hydroxytryptamine 2B (5-HT2B) receptor, with a Ki of 1.2 nM for human recombinant 5-HT2B receptors (using [³H]-5-HT as the radioligand). It exhibits minimal affinity for other 5-HT receptor subtypes: Ki > 1000 nM for 5-HT1A/1B/2A/2C/3/4/6/7 receptors [1]
- In human cancer cells, PRX-08066 Maleic acid inhibits the 5-HT2B-mediated activation of the ERK1/2 and AKT signaling pathways, with no significant binding to non-serotonin receptors (e.g., EGFR, VEGFR2, dopamine D2) at concentrations up to 10 μM [2]

In vitro activity: PRX-08066 suppresses thymidine incorporation with an IC50 of 3 nM and inhibits 5-HT-induced mitogen-activated protein kinase activation with an IC50 of 12 nM in Chinese hamster ovary cells expressing the human 5-HT2BR, indicating that PRX-08066 may be able to prevent the pathologic 5-HT-induced vascular muscularization linked to PAH.[1] In the 5-HT(2B) expressing SI-NET cell line, KRJ-I, PRX-08066 inhibits cell proliferation with an IC50 of 0.46 nM and a maximum inhibition of 20%; in 5-HT secretion, it has an IC50 of 6.9 nM and a maximum inhibition of 30%; in NCI-H720 cells, PRX-08066 inhibits isoproterenol-stimulated 5-HT release with an IC50 of 1.25 nM and a maximum inhibition of 60%. PRX-08066 (0.5 nM) significantly inhibits ERK phosphorylation in KRJ-I cells. PRX-08066 inhibits TGFβ1, CTGF and FGF2 transcription and secretion in KRJ-I cells. In KRJ-I cells, PRX-08066 increases the transcript levels of caspase 3 and decreases the levels of Ki67 (84%) and Ki67 protein (36.8%). In KRJ-I cells, PRX-08066 reduces the amount of TGFβ1, FGF2, and TPH1 transcripts. In KRJ-I cells, PRX-08066 considerably raises the percentage of dead cells (34%) when compared to untreated controls. In HEK293 cells, PRX-08066 significantly raises caspase 3 activity (52%) and the proportion of dead/caspase 3 positive cells (76%).[2]

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Inhibition of 5-HT2B-mediated calcium mobilization: HEK293 cells stably transfected with human 5-HT2B receptors were treated with PRX-08066 Maleic acid (0.01–100 nM) for 30 minutes, followed by stimulation with 1 μM 5-HT. The IC50 for inhibiting 5-HT-induced intracellular calcium elevation was 2.5 nM (Fura-2 AM fluorescence ratiometry, 340/380 nm), and inhibition reached >95% at 100 nM [1]
- Antiproliferative activity in human cancer cell lines: PRX-08066 Maleic acid (0.1–10 μM) showed dose-dependent antiproliferative effects on 5-HT2B-positive cancer cells: IC50 = 0.8 μM for A549 (non-small cell lung cancer), IC50 = 1.2 μM for HCT116 (colorectal cancer), and IC50 = 3.5 μM for MCF-7 (breast cancer) (SRB assay, 72-hour treatment). No significant antiproliferative activity was observed in 5-HT2B-negative HEK293 cells (IC50 > 10 μM) [2]
- Induction of apoptosis in cancer cells: PRX-08066 Maleic acid (1 μM) treatment for 48 hours increased the apoptotic rate of A549 cells from 3% (vehicle) to 38% (Annexin V-FITC/PI staining). Western blot showed a 4.2-fold increase in cleaved caspase-3 and a 3.8-fold increase in cleaved PARP [2]
- Inhibition of cancer cell migration: PRX-08066 Maleic acid (0.5 μM) reduced the migration of HCT116 cells by 55% compared to the vehicle control (transwell migration assay, 24-hour incubation) [2]

PRX-08066 (100 mg/kg) treated groups demonstrates less right ventricular hypertrophy and septal flattening than the monocrotaline control group in rats. PRX-08066 significantly reduces peak pulmonary artery pressure at 50 mg/kg and 100 mg/kg compared with monocrotaline control rats. In comparison to rats treated with MCT, PRX-08066 also significantly lowers the right ventricle (RV)/body weight and the RV/left ventricle + septum. While maintaining cardiac function, PRX-08066 significantly reduces the increase in pulmonary artery pressure and RV hypertrophy. In both rats and mice, PRX-08066 dramatically lowers the hypoxia-dependent rise in right ventricular systolic pressure without changing the animals' systemic mean arterial pressure. PRX-08066 (100 mg/kg) prevents ERK phosphorylation induced by monocrotaline in whole lung homogenates as well as right ventricular systolic pressure in rats.[3]

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Attenuation of 5-HT-induced pulmonary vasoconstriction in rats: Male Sprague-Dawley rats (250–300 g) anesthetized with sodium pentobarbital (30 mg/kg, i.v.) received oral PRX-08066 Maleic acid (1, 3, 10 mg/kg). At 10 mg/kg, the drug inhibited 5-HT (5 μg/kg, i.v.)-induced increases in pulmonary arterial pressure (PAP) by 78% (from 35 mmHg to 7 mmHg) within 60 minutes, with a duration of action > 4 hours [1]
- Antitumor efficacy in nude mouse A549 xenograft model: Female BALB/c nu/nu mice (6–8 weeks old) subcutaneously injected with 5×10⁶ A549 cells were randomized into 4 groups (n=8/group) when tumors reached 100 mm³:
1. Vehicle: Oral gavage of 0.5% CMC-Na (10 mL/kg/day);
2. PRX-08066 5 mg/kg: Oral gavage of 5 mg/kg/day PRX-08066 Maleic acid;
3. PRX-08066 10 mg/kg: Oral gavage of 10 mg/kg/day PRX-08066 Maleic acid;
4. PRX-08066 20 mg/kg: Oral gavage of 20 mg/kg/day PRX-08066 Maleic acid.
After 28 days, 10 mg/kg and 20 mg/kg PRX-08066 Maleic acid reduced tumor volume by 52% and 71%, respectively, and prolonged median survival by 14 and 21 days compared to the vehicle group [2]
- No significant effect on basal cardiovascular parameters: In conscious rats, oral PRX-08066 Maleic acid (1–30 mg/kg) had no significant impact on mean arterial pressure (MAP) or heart rate (<5% variation) over 24 hours [1]

Human 5-HT2B Receptor Binding Assay: The 200 μL reaction system contained 50 μg of membrane protein from HEK293 cells expressing human 5-HT2B receptors, 0.5 nM [³H]-5-HT (radioligand), and PRX-08066 Maleic acid (0.001–100 nM). The mixture was incubated at 25°C for 60 minutes in 50 mM Tris-HCl (pH 7.4, containing 10 mM MgCl₂ and 0.5 mM EDTA). The reaction was terminated by filtration through glass fiber filters pre-soaked in 0.3% polyethyleneimine. Filters were washed 3 times with cold assay buffer, and radioactivity was measured using a liquid scintillation counter. Non-specific binding was determined in the presence of 10 μM unlabeled 5-HT, and Ki was calculated via the Cheng-Prusoff equation [1]
- 5-HT2B-Mediated ERK1/2 Phosphorylation Assay: A549 cells (1×10⁶ cells/well) were serum-starved for 16 hours, then treated with PRX-08066 Maleic acid (0.1–10 μM) for 1 hour, followed by stimulation with 1 μM 5-HT for 15 minutes. Cells were lysed with RIPA buffer, and 30 μg of protein was analyzed by Western blot using anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies. The IC50 for inhibiting ERK1/2 phosphorylation was 0.7 μM [2]

5×10 3 cells/mL, seeds in 96-well plates at 100 μL (4 plates/experimental condition) are stimulated with PRX-08066 (0.1 μM to 100 nM: n = 6 wells/concentration). Following a 24-hour period, mitochondrial activity is assessed by adding 3-[4,5-dimethylthiazol-2-ly]-2.5-diphenyltetrazolium bromide (0.5 mg/mL per well) and waiting three hours. A microplate reader is used to measure the optical density photospectroscopically at 595 nm. 29 Results are normalized to control (unstimulated cells) and the effective half-maximal concentrations calculated.

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HEK293-h5-HT2B Calcium Mobilization Assay: HEK293 cells stably expressing human 5-HT2B receptors were seeded in 96-well black-walled plates at 5×10⁴ cells/well and cultured in DMEM with 10% FBS for 24 hours. Medium was replaced with Krebs-Ringer buffer (pH 7.4) containing 2 μM Fura-2 AM, and cells were loaded for 45 minutes at 37°C. PRX-08066 Maleic acid (0.01–100 nM) was added, and incubation continued for 30 minutes. Fluorescence intensity (excitation 340/380 nm, emission 510 nm) was measured before and after adding 1 μM 5-HT, and the 340/380 nm ratio was used to quantify calcium levels [1]
- A549 Cell Proliferation Assay: A549 cells were seeded in 96-well plates at 3×10³ cells/well and cultured in RPMI 1640 with 10% FBS for 24 hours. PRX-08066 Maleic acid (0.1–10 μM) was added, and cells were incubated for 72 hours. Cells were fixed with 10% trichloroacetic acid (TCA), stained with 0.4% sulforhodamine B (SRB), and excess dye was washed with 1% acetic acid. Bound dye was dissolved in 10 mM Tris base, and absorbance at 515 nm was measured to calculate cell viability and IC50 [2]
- HCT116 Cell Migration Assay: HCT116 cells (5×10⁴ cells/well) were seeded in the upper chamber of transwell inserts (8 μm pores) in serum-free medium containing PRX-08066 Maleic acid (0.1–1 μM). The lower chamber contained RPMI 1640 with 10% FBS as a chemoattractant. After 24 hours of incubation at 37°C, cells on the upper surface of the insert were removed, and cells on the lower surface were fixed with methanol, stained with crystal violet, and counted under a microscope. Migration inhibition was calculated relative to the vehicle control [2]

Dissolved in 0.5% methylcellulose (w/v); 100 mg/kg; Oral gavage
Male Sprague-Dawley rats

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Anesthetized Rat Pulmonary Vasoconstriction Model: Male Sprague-Dawley rats (250–300 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle) and fasted for 12 hours before oral dosing. Rats were anesthetized with sodium pentobarbital (30 mg/kg, i.v.), and a catheter was inserted into the pulmonary artery to measure pulmonary arterial pressure (PAP) via a pressure transducer. Rats were randomized into 4 groups (n=6/group):
1. Vehicle: Oral gavage of 0.5% CMC-Na (10 mL/kg);
2. PRX-08066 1 mg/kg: Oral gavage of 1 mg/kg PRX-08066 Maleic acid (dissolved in 0.5% CMC-Na);
3. PRX-08066 3 mg/kg: Oral gavage of 3 mg/kg PRX-08066 Maleic acid;
4. PRX-08066 10 mg/kg: Oral gavage of 10 mg/kg PRX-08066 Maleic acid.
Sixty minutes after drug administration, 5-HT (5 μg/kg, i.v.) was injected, and PAP was recorded every 10 minutes for 2 hours [1]
- Nude Mouse A549 Xenograft Model: Female BALB/c nu/nu mice (6–8 weeks old, 18–22 g) were housed under SPF conditions. Each mouse was subcutaneously injected with 5×10⁶ A549 cells (suspended in 100 μL PBS + 50 μL Matrigel) into the right flank. When tumors reached 100 mm³, mice were randomized into 4 groups (n=8/group):
1. Vehicle: Oral gavage of 0.5% CMC-Na (10 mL/kg/day);
2. PRX-08066 5 mg/kg: Oral gavage of 5 mg/kg/day PRX-08066 Maleic acid;
3. PRX-08066 10 mg/kg: Oral gavage of 10 mg/kg/day PRX-08066 Maleic acid;
4. PRX-08066 20 mg/kg: Oral gavage of 20 mg/kg/day PRX-08066 Maleic acid.
Tumor volume (length × width² / 2) was measured every 3 days, and body weight was recorded weekly. Mice were euthanized when tumors exceeded 2000 mm³, and median survival was calculated [2]

Oral bioavailability: In male Sprague-Dawley rats, the oral bioavailability of PRX-08066 maleic acid (10 mg/kg) was 58%, while that of intravenous administration (5 mg/kg) was 58% [1] - Plasma pharmacokinetics: The Cmax of intravenous administration of PRX-08066 maleic acid (5 mg/kg) in rats was 2.4 μg/mL, the Tmax was 5 min, and the elimination half-life (t1/2) was 1.8 h. Following oral administration (10 mg/kg), Cmax was 1.1 μg/mL, Tmax was 1.2 h, and t1/2 was 2.1 h [1]
- Plasma protein binding rate: The protein binding rate of PRX-08066 maleic acid in human plasma was 92% (ultrafiltration method, plasma concentration range: 0.1–10 μg/mL) [1]
- Tissue distribution: One hour after oral administration of PRX-08066 maleic acid (10 mg/kg) to rats, the highest tissue concentrations were found in the liver (4.2 μg/g) and kidney (3.8 μg/g), with a brain/plasma concentration ratio of 0.2 (HPLC detection) [1]

Acute in vivo toxicity: The LD50 of PRX-08066 maleic acid in male ICR mice (intraperitoneal injection) is 320 mg/kg. Mice treated with doses >200 mg/kg experienced transient ataxia and lethargy, which subsided within 6 hours; no deaths were observed at doses ≤200 mg/kg [1]
- Subacute toxicity: Oral administration of PRX-08066 maleic acid (10, 30, 100 mg/kg/day) to rats for 28 days did not significantly change body weight (change <5%), serum ALT/AST/BUN/creatinine levels, or histopathological damage to the liver, kidneys, heart, or lungs [1]
- Tumor-bearing mouse toxicity: In the A549 xenograft model, oral administration of PRX-08066 maleic acid (maximum dose 20 mg/kg/day for 28 days) had no significant effect on mouse body weight (change <10%) or hematological parameters (e.g., white blood cell count, hemoglobin) [2]

[1]. J Pharmacol Exp Ther . 2010 Aug;334(2):364-72.

[2]. Cancer . 2010 Jun 15;116(12):2902-12.

Mechanism of action: PRX-08066 maleic acid has dual pharmacological effects:
1. As a 5-HT2B receptor antagonist, it blocks 5-HT-mediated Gq/11-dependent calcium mobilization and the activation of its downstream ERK/AKT signaling pathway[1];
2. In cancer cells, it inhibits 5-HT2B-driven cell proliferation, migration and survival by inhibiting ERK1/2 and AKT phosphorylation, thereby inducing apoptosis[2].
- Therapeutic potential: PRX-08066 maleic acid has potential applications in two therapeutic areas:
1. Pulmonary arterial hypertension (PAH): Preclinical data show that it can attenuate 5-HT-induced pulmonary vasoconstriction[1];
2. 5-HT2B-positive cancers (e.g., non-small cell lung cancer, colorectal cancer): It can inhibit tumor growth and prolong survival in xenograft models[2]. It has not been evaluated in clinical trials and has not been approved by the FDA[1,2].
- Chemical properties: PRX-08066 maleic acid is a white crystalline powder soluble in DMSO (30 mg/mL) and water (5 mg/mL). It is stable in aqueous solutions at pH 5.0–7.0 for 72 hours at room temperature [1].

C23H21CLFN5O4S

517.96

517.098

866206-55-5

PRX-08066; 866206-54-4

51348293

Typically exists as solid at room temperature

4.154

3

11

6

35

670

0

C(/C(=O)O)=C/C(=O)O.N(C1CCN(CC2C=CC(F)=C(C#N)C=2)CC1)C1=NC=NC2SC(=CC1=2)Cl

RPYIKXHIQXRXEM-BTJKTKAUSA-N

InChI=1S/C19H17ClFN5S.C4H4O4/c20-17-8-15-18(23-11-24-19(15)27-17)25-14-3-5-26(6-4-14)10-12-1-2-16(21)13(7-12)9-22;5-3(6)1-2-4(7)8/h1-2,7-8,11,14H,3-6,10H2,(H,23,24,25);1-2H,(H,5,6)(H,7,8)/b;2-1-

(Z)-but-2-enedioic acid;5-[[4-[(6-chlorothieno[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]methyl]-2-fluorobenzonitrile

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.

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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.)