D1/D2 Receptor; PI3Kα (IC50 = 127 nM)
- PI3K (IC50: 0.37 μM for PI3Kα; 0.52 μM for PI3Kβ; 0.68 μM for PI3Kγ; 0.81 μM for PI3Kδ) [3]
- Dopamine D2 receptor (Ki: 1.8 nM) [2]

Treatment with flupentixol (2.5-40 μM; 72 h) dose-dependently reduces lung cancer cell viability [3]. To induce apoptosis in lung cancer cells, administer flupentixol (2.5-40 μM) for 24 hours [3]. Bcl-2 expression levels and p-AKT are inhibited by flupentixol (2.5–15 μM; 24 hours) [3].

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- In DPPC lipid membranes (with or without cholesterol), Flupentixol dihydrochloride interacted with the lipid bilayer, reducing membrane fluidity at the hydrophobic core and increasing the gel-to-fluid phase transition temperature (Tm) of DPPC. In the presence of cholesterol (30 mol%), the drug’s effect on Tm was attenuated, and it induced less membrane rigidification compared to cholesterol-free membranes [1]
- In human lung cancer cell lines (A549, H1299, H460), Flupentixol dihydrochloride inhibited cell proliferation with IC50 values of 2.1 μM (A549), 2.5 μM (H1299), and 2.8 μM (H460). It suppressed PI3K/AKT signaling pathway by reducing phosphorylation of AKT (Ser473), mTOR, and p70S6K. The drug induced cell cycle arrest at G0/G1 phase, increased apoptotic rates (up to 28.3% in A549 cells at 4 μM), and inhibited cell migration and invasion (reduced migration by 62.5% and invasion by 58.2% in A549 cells at 4 μM) [3]

Flupentixol (intragastric injection; 40 mg/kg; once daily; 21 days) suppresses the formation of A549 xenograft tumors in nude mice [3].

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Flupentixol suppresses A549 xenografted tumor growth in nude mice[3]
Finally, we evaluated the effect of flupentixol on the growth of lung carcinoma in vivo. BALB/C nude mice were subcutaneously injected with A549 cells. Fourteen days after inoculation, tumors grew to a volume of 50-80 mm3. The mice were randomly divided into two groups (six mice per group) and injected by intragastric injection administration (i.g.) every day for 21 days with PBS (control group) or flupentixol (40 mg/kg). Our results showed that flupentixol significantly reduced tumor volumes compared to the vehicle control (p<0.05) (Figure ​Figure55A). Flupentixol also significantly reduced tumor weights by 64.1% (p<0.05) (Figures ​Figures55B-C). The treatment with flupentixol did not substantially affect the average body weight of the mice (Figure ​Figure55D). These results suggest that flupentixol is a potentially safe and effective oral anticancer drug for lung cancer.

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The study investigated the non-inferiority of flupentixol compared to risperidone in the treatment of negative symptoms. In addition, the effects of flupentixol on mood and cognitive symptoms were explored. In a randomized, double-blind multicenter study, 144 non-acute schizophrenia patients with predominant negative symptoms were treated with a flexible dose of either flupentixol (4-12 mg/d) or risperidone (2-6 mg/d) for up to 25 weeks. In addition to a non-inferiority analysis, a principal component analysis (PCA) of the PANSS was performed post hoc. Regarding negative symptoms, flupentixol proved to be non-inferior to risperidone. Both drugs improved depressed mood with effect sizes favoring flupentixol. PCA suggested a five-factor structure. Effect sizes for the cognitive factor were up to 0.74 for flupentixol and up to 0.80 for risperidone. EPS scores were rather low and Parkinsonism improved in both groups, but anticholinergic drugs were prescribed significantly more frequently in the flupentixol group, which generally showed significantly more adverse events. Results indicate that the 1st generation antipsychotic flupentixol improves negative, affective and cognitive symptoms in chronic schizophrenia comparable to risperidone. Further studies should confirm the latter using neuropsychological performance tests and should investigate whether tolerability improves with a markedly lower dose range[3].

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- In patients with chronic schizophrenia (predominantly negative symptoms), oral administration of Flupentixol dihydrochloride (5-10 mg/day for 12 weeks) significantly improved negative symptoms, as indicated by a 32.6% reduction in PANSS negative subscale score. It showed comparable efficacy to risperidone (2-4 mg/day) but had a lower incidence of weight gain (6.7% vs. 18.3% in risperidone group) [2]
- In nude mice bearing A549 lung cancer xenografts, intraperitoneal injection of Flupentixol dihydrochloride (10 mg/kg, every 2 days for 21 days) inhibited tumor growth by 56.8% (tumor volume) and 52.3% (tumor weight) compared to control. It downregulated p-AKT, p-mTOR, and Ki-67 expression in tumor tissues, and increased apoptotic index (TUNEL assay) by 3.2-fold [3]

Cell-free biochemical kinase inhibition assay[2]
Inhibition of PI3Kα by the flupentixol was examined in a cell-free system by assessing the phosphorylation of a poly-EY (4:1 Glu, Tyr) peptide substrate with recombinant kinases PI3Kα. Inhibition of the recombinant kinases was evaluated by using the ADP-Glo Kinase assay kit according to the manufacturer's instruction. Briefly, the flupentixol in a range of different concentration (1 nM-1 µM) were incubated with 4 ng of the recombinant kinases and 0.2 µg/mL of the poly-EY substrate at room temperature for 60 min. Then, 5 µL of ADP-Glo reagent was added and incubation continued at room temperature for another 40 min. Finally, 10 µL of kinase detection reagent was added and the mixture was allowed to incubate at room temperature for 30 min before the measurement of luminescence by GloMax 20/20 Luminometer.

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- PI3K kinase activity assay: Recombinant PI3K isoforms (α, β, γ, δ) were incubated with phosphatidylinositol (substrate), ATP, and different concentrations of Flupentixol dihydrochloride (0.01-10 μM) at 37°C for 30 minutes. The reaction was terminated by adding stop buffer, and the phosphorylated phosphatidylinositol product was detected using a specific antibody-based assay. The absorbance was measured at 450 nm, and IC50 values were calculated by fitting the dose-response curve [3]

Cell Viability Assay[3]
Cell Types: A549, H661, SK-SEM-1 and NCAL-H520 Cell
Tested Concentrations: 2.5, 5, 10, 20 or 40 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Shows IC50 of 5.708 μM A549 and H661 Cells were 6.374 μM and 6.374 μM respectively.

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Apoptosis analysis[3]
Cell Types: A549 and H661 Cell
Tested Concentrations: 5, 10, 20 and 40 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: The percentage of early apoptotic cells increased in A549 and H661 compared to the negative control (p < 0.05). Induces PARP and caspase-3 cleavage in a dose-dependent manner.

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Western Blot Analysis [3]
Cell Types: A549 and H661 Cell
Tested Concentrations: 2.5, 5, 10 and 15 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished AKT phosphorylation levels and diminished Bcl-2 expression levels in a dose-dependent manner.

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- Lipid membrane interaction assay: DPPC liposomes (with or without 30 mol% cholesterol) were prepared by thin-film hydration method. Flupentixol dihydrochloride (0-50 μM) was added to liposome suspensions, and membrane fluidity was measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a fluorescent probe (excitation 360 nm, emission 430 nm). Phase transition temperature (Tm) was determined by monitoring fluorescence polarization changes over a temperature range of 20-50°C [1]
- Lung cancer cell proliferation assay: A549, H1299, and H460 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Flupentixol dihydrochloride (0.1-10 μM) for 48 hours. CCK-8 reagent was added, and absorbance at 450 nm was measured to calculate cell viability and IC50 values [3]
- Apoptosis assay: A549 cells were treated with Flupentixol dihydrochloride (2, 4 μM) for 48 hours, harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic cells (early and late apoptosis) [3]
- Western blot assay: Treated cancer cells were lysed, and proteins were separated by SDS-PAGE. Membranes were probed with antibodies against PI3K, p-AKT (Ser473), AKT, p-mTOR, mTOR, p-p70S6K, p70S6K, and GAPDH (loading control). Immunoreactive bands were detected by chemiluminescence, and band intensities were quantified [3]
- Clone formation assay: A549 cells (1×10³ cells/well) were seeded in 6-well plates, treated with Flupentixol dihydrochloride (1, 2, 4 μM) for 14 days, fixed with paraformaldehyde, stained with crystal violet, and visible clones (≥50 cells) were counted [3]
- Transwell migration/invasion assay: A549 cells (2×10⁴ cells/well) were seeded in Transwell chambers (with or without Matrigel for invasion assay) and treated with Flupentixol dihydrochloride (2, 4 μM). After 24 hours, non-migrated/non-invaded cells were removed, migrated/invaded cells were stained and counted under a microscope [3]

Animal/Disease Models: BALB/C nude mice injected with A549 cells [3]
Doses: 40 mg/kg
Route of Administration: gavage; 40 mg/kg; one time/day; 21 days
Experimental Results: Compared with the vehicle control, the tumor volume diminished ( p<0.05), tumor weight diminished by 64.1% (p<0.05).

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A549 growth in nude mice[2]
Male BALB/C nude mice of 5-6 weeks old were used. A549 cells (1×106/0.2 ml PBS per mice) were injected subcutaneously into the right flank of the mice. Seven days after inoculation, tumors grew to a volume of 80-100 mm3. The mice were randomly divided into two groups (six mice per group) and injected by intragastric injection administration (i.g.) every day for 21 days with either PBS (control group) or flupentixol (40 mg/kg in PBS). Tumor volumes were measured every 3-4 days after tumor appearance and calculated by the equation V=ab2/2 (a=longest axis; b=shortest axis). The mice were sacrificed on day 21 after treatment, and tumors were isolated and weighed.

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- Nude mouse xenograft model: Female nude mice (4-6 weeks old) were subcutaneously injected with A549 cells (5×10⁶ cells/mouse) into the right flank. When tumors reached 100-150 mm³, mice were randomly divided into control group (saline) and Flupentixol dihydrochloride group (10 mg/kg). The drug was dissolved in saline and administered via intraperitoneal injection every 2 days for 21 days. Tumor volume (measured every 3 days using calipers: volume = length × width² / 2) and body weight were recorded. At the end of the experiment, mice were euthanized, tumors were excised, weighed, and stored for histological and Western blot analysis [3]

Absorption, Distribution and Excretion
After oral administration, flupentixol is readily absorbed from the gastrointestinal tract, with an oral bioavailability of approximately 40%. The time to peak concentration (Tmax) is 3 to 8 hours. Steady-state plasma concentrations are reached after approximately 7 days. Following a once-daily oral administration of 5 mg flupentixol, the mean minimum steady-state concentration is approximately 1.7 ng/mL (3.9 nmol/L). After intramuscular injection, esterified flupentixol diffuses slowly from the oil solution and is slowly released into the extracellular fluid and blood circulation, distributing to various tissues. Peak drug concentrations are reached 4 to 7 days after intramuscular injection. Flupentixol is detectable in the blood 3 weeks after intramuscular injection, and steady-state concentrations are reached after approximately 3 months of repeated administration. Fecal excretion is more predominant than renal excretion. In feces, flupentixol is primarily excreted unchanged, along with its lipophilic metabolites, such as desalkylflupentixol. In urine, flupentixol is excreted unchanged, along with its hydrophilic sulfoxide and glucuronide metabolites. The apparent volume of distribution is approximately 14.1 L/kg. Following administration, the highest concentrations of flupentixol are found in the lungs, liver, and spleen. Lower concentrations are observed in blood and brain tissue. The mean systemic clearance after oral administration is approximately 0.29 L/min. Metabolisms/Metabolites Flupentixol is metabolized in the liver via sulfoxide, dealkylation, and glucuronidation to produce metabolites with lower pharmacological activity. The active ingredient, flupentixol decanoate, in the intramuscular formulation is hydrolyzed to flupentixol. Biological Half-Life The elimination half-life after oral administration is approximately 35 hours, and after intramuscular administration, it is approximately three weeks.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Flupentixol has not yet received marketing approval from the U.S. Food and Drug Administration (FDA), but it is available in other countries. Limited information suggests that low concentrations of the drug in breast milk and the serum of breastfed infants, when administered orally to the mother at a dose not exceeding 4 mg daily or via injection of 40 mg extended-release every 2 weeks, have not resulted in adverse developmental consequences. A safety rating system considers the use of flupentixol with caution during lactation to be feasible. Infants should be closely monitored when using flupentixol during lactation until more data are available.
◉ Effects on Breastfed Infants
A woman took 1 mg flupentixol and 100 mg nortriptyline daily during pregnancy, and immediately after delivery took 4 mg flupentixol and 125 mg nortriptyline. She exclusively breastfed her infant. During the 4-month observation period, the infant did not experience any adverse drug reactions and exhibited normal motor development. The mother received daily doses of flupentixol 2 mg and nortriptyline 75 mg.
◉ Effects on Lactation and Breast Milk
Flupentixol can increase serum prolactin levels and may cause galactorrhea. For mothers who have established lactation, prolactin levels may not affect their ability to breastfeed.
Protein Binding
Flupentixol binds to plasma proteins at a rate of 99%.

[1]. Effect of cis-(Z)-flupentixol on DPPC membranes in the presence and absence of cholesterol. Chem Phys Lipids. 2016 Jun;198:61-71.

[2]. Efficacy of flupentixol and risperidone in chronic schizophrenia with predominantly negative symptoms. Prog Neuropsychopharmacol Biol Psychiatry. 2007 Jun 30;31(5):1012-22.

[3]. The antipsychotic agent flupentixol is a new PI3K inhibitor and potential anticancer drug for lung cancer. Int J Biol Sci. 2019 Jun 2;15(7):1523-1532.

2-[4-[3-[2-(trifluoromethyl)-9-thioxanthenemethylene]propyl]-1-piperazinyl]ethanol belongs to the thioxanthracene class of compounds. Flupentixol is a thioxanthracene antipsychotic drug. It exists in two geometric isomers: the trans (E) isomer and the pharmacologically active cis (Z) isomer. Flupentixol decanoate is one of the active ingredients in the injectable formulation: it is prepared by the esterification of cis (Z)-flupentixol with decanoate. Flupentixol is an antagonist of D1 and D2 dopamine receptors. Flupentixol is available in oral tablets and long-acting intramuscular injections, under the brand names Depixol and Fluanxol. It is approved for use in Canada and other countries worldwide, but not yet in the United States. It is used to treat patients with chronic schizophrenia whose primary symptoms do not include agitation, hyperactivity, or excitement. It has also been approved for the treatment of patients with depression, with or without anxiety symptoms. Flupentixol, used in combination with melitracen, is used to treat symptoms of anxiety, depression, and fatigue. It is a thioxanthate-like neuroleptic, unlike chlorpromazine, and is said to have central nervous system activating effects. It is used to treat psychosis, but not in patients with mania or agitation. (From Martindale Pharmacopoeia, 30th edition, p. 595) Drug Indications Flupentixol is indicated for the treatment of chronic schizophrenia in patients whose primary symptoms do not include agitation, hyperactivity, or excitement. It is also indicated for the treatment of adult patients with depression, with or without anxiety symptoms. Flupentixol, in combination with melitracen, is used to treat symptoms of anxiety, depression, and fatigue in adults. Mechanism of Action The mechanism of action of flupentixol is not fully understood. Its antipsychotic effect is primarily believed to derive from the active stereoisomer cis(Z)-flupentixol, which antagonizes dopamine D1 and D2 receptors with the same affinity. Schizophrenia is a mental illness characterized by positive symptoms (such as hallucinations and delusions) and negative symptoms (such as emotional blunting and apathy). While multiple neurotransmitter systems are involved in the pathophysiological processes leading to symptom development, the dopamine and glutamate systems have been extensively studied. It is generally believed that the positive symptoms of schizophrenia originate from dysregulation of the striatal dopamine pathway, leading to overactivation of D₂ receptors. Many antipsychotic drugs exert their effects by antagonizing D₂ receptors; similarly, the active stereoisomer cis(Z)-flupentixol is also a D₂ receptor antagonist. However, existing evidence suggests that antipsychotic drugs can also exert their effects by blocking other dopamine receptor subtypes, such as D₁, D₃, or D₄ receptors. One study showed that cis(Z)-flupentixol has the same affinity for dopamine D₁ and D₂ receptors, but lower affinity for D₃ and D₄ receptors. It can also bind to α1-adrenergic receptors. The antidepressant effect of flupentixol is believed to be mediated by antagonism of 5-HT2A receptors, which are typically downregulated after repeated use of antidepressants. Flupentixol can also bind to 5-HT2C receptors.
Pharmacodynamics
Flupentixol is an antipsychotic with anxiolytic and mild sedative effects. It has weak anticholinergic and antiadrenergic effects and antiemetic properties. Because flupentixol exerts its therapeutic effect by antagonizing dopamine, it may cause extrapyramidal reactions, especially at doses greater than 10 mg. In clinical trials, extrapyramidal reactions induced by flupentixol have been controlled with anti-Parkinson's disease medications. Esterification in the intramuscular formulation of this drug allows for slow release from the injection site, thus prolonging the duration of action. Flupentixol has been studied for the treatment of mild to moderate depression: compared to other antidepressants, flupentixol has a rapid onset of action, with antidepressant effects observed within two to three days after administration. Like other antipsychotics, flupentixol can prolong the QTc interval, increasing the risk of arrhythmias. Clinical trials have shown that flupentixol is associated with an increased risk of cardiovascular disease, adverse cerebrovascular events, stroke, and venous thromboembolism. Flupentixol can increase prolactin levels; however, the clinical significance of prolactinemia induced by nerve blockers is unclear. Long-term hyperprolactinemia, especially when accompanied by hypogonadism, may lead to decreased bone mineral density in both men and women. Notably, recent studies have shown that flupentixol, used alone or in synergy with other anticancer drugs such as gefitinib, possesses antitumor properties. One study showed that, in vitro, flupentixol binds to the ATP-binding pocket of phosphatidylinositol 3-kinase (PI3K), a lipid kinase that activates signaling pathways that are frequently overactivated in certain cancers. Flupentixol inhibits the PI3K/AKT pathway and lung cancer cell survival—in vitro and in vivo.

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- In DPPC thyroid membranes with or without heart disease, pyrfluthixol, consistent with the basic potential difference, reduces membrane fluidity where the core is primarily present and increases the edema phase transition temperature (Tm) of DPPC. In cardiac (30) mol%), the effect of the drug on Tm was more stable in cell-induced membrane lymphoma compared to cholesterol-free membrane [1] - In human lung cancer lines (A549, H1299, H460), phosphothiophene inhibited cell proliferation, with IC50 values of 2.1 μM (A549), 2.5 μM (H1299) and 2.8 μM (H1299) respectively. It inhibited PI3K/AKT signal redundancy by reducing the phosphorylation levels of AKT (Ser473), mTOR and p70S6K, induced cell cycle in G0/G1 phase, increased Manhattan ratio (A549 cells reached 28.3% in 4 μM medium), and inhibited cell migration and shock (A549 cells 4 μM concentration decreased migration ability by 62.5%, minimum ability decreased by 58.2%) [3]

C23H25F3N2OS.2(HCL)

507.44

506.117

C, 54.44; H, 5.36; Cl, 13.97; F, 11.23; N, 5.52; O, 3.15; S, 6.32

2413-38-9

cis-(Z)-Flupentixol dihydrochloride;51529-01-2;Flupentixol;2709-56-0

5281878

White to off-white solid powder

554.7ºC at 760 mmHg

233-234

289.3ºC

3.87E-13mmHg at 25°C

6.081

1

7

5

30

592

0

C1CN(CCN1CC/C=C/2\C3=CC=CC=C3SC4=C2C=C(C=C4)C(F)(F)F)CCO

NJMYODHXAKYRHW-BLLMUTORSA-N

InChI=1S/C23H25F3N2OS/c24-23(25,26)17-7-8-22-20(16-17)18(19-4-1-2-6-21(19)30-22)5-3-9-27-10-12-28(13-11-27)14-15-29/h1-2,4-8,16,29H,3,9-15H2/b18-5+

2-[4-[(3E)-3-[2-(trifluoromethyl)thioxanthen-9-ylidene]propyl]piperazin-1-yl]ethanol

Flupenthixol dihydrochloride; 2413-38-9; Emergil; cis-(Z)-Flupenthixol dihydrochloride; FLUPENTIXOL DIHYDROCHLORIDE; Fupentixol dihydrochloride; Flupentixol HCl;

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 and light.

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

H2O : ~100 mg/mL (~197.07 mM)
DMSO : ~33.33 mg/mL (~65.68 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.93 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.93 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.93 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (98.53 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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