D1/D2 Receptor; PI3Kα (IC50 = 127 nM)

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].

Intranastric injections of flupentixol (40 mg/kg, once daily for 21 days) prevent the formation of A549 xenograft tumors in mice that are not clothed [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].

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.

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: H661 and A549 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.

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.

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 plasma 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 approximately 3 months after repeated administration. Fecal excretion is more common 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. Metabolites/Metabolites: Flupentixol is metabolized in the liver via sulfoxide formation, dealkylation, and glucuronidation to form pharmacologically inactive metabolites. In the intramuscular formulation, the active ingredient, flupentixol decanoate, is hydrolyzed to flupentixol. Biochemical 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 flupentixol in breast milk and the serum of breastfed infants, even with a maternal oral dose not exceeding 4 mg daily or an injectable 40 mg extended-release dose 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 postpartum took 4 mg flupentixol and 125 mg nortriptyline daily. 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.

Cis-flupentixol is a flupentixol compound with a cis configuration. It is a dopaminergic antagonist and the conjugate base of cis-flupentixol(2+). Flupentixol is a thiol-type antipsychotic drug. It exists in two geometric isomers: trans (E) and the pharmacologically active cis (Z) isomer. Flupentixol decanoate is one of the active ingredients in the injectable formulation, prepared by esterification of cis (Z)-flupentixol with decanoate. Flupentixol is an antagonist of both D1 and D2 dopamine receptors. Flupentixol is available in oral tablets and long-acting intramuscular injections under brand names including Depixol and Fluanxol. It is approved for use in Canada and other countries worldwide, but not in the United States. It is used to treat chronic schizophrenia in patients whose primary symptoms do not include agitation, hyperactivity, or excitement. It has also been approved for the treatment of depressive symptoms, with or without anxiety. Flupentixol, used in combination with melitracen, is used to treat symptoms of anxiety, depression, and fatigue. Flupentixol is a thiol-based neuroleptic, unlike chlorpromazine, and is claimed to have central nervous system activating effects. It is used to treat psychosis, but not in patients with mania or agitation. (Excerpt 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 indicated for the treatment of adult patients with depression, with or without anxiety symptoms. Flupentixol, used in combination with melitracen, is indicated for the treatment of adult symptoms of anxiety, depression, and fatigue. 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. An in vitro study demonstrated that flupentixol binds to the ATP-binding pocket of phosphatidylinositol 3-kinase (PI3K), a lipid kinase that activates signaling pathways frequently overactivated in certain cancers. Flupentixol inhibits the PI3K/AKT pathway and lung cancer cell survival both in vitro and in vivo.

C23H25N2OF3S

434.5176

434.164

C, 63.58; H, 5.80; F, 13.12; N, 6.45; O, 3.68; S, 7.38

2709-56-0

Flupentixol dihydrochloride;2413-38-9;cis-(Z)-Flupentixol dihydrochloride;51529-01-2; 51529-02-3 [(E)-Flupentixol]

5281881

Typically exists as solid at room temperature

1.306g/cm3

554.7ºC at 760mmHg

233-234

289.3ºC

1.607

4.477

1

7

5

30

592

0

OCCN1CCN(CC/C=C2\C3=CC=CC=C3SC3C=CC(=CC\2=3)C(F)(F)F)CC1

NJMYODHXAKYRHW-DVZOWYKESA-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-[(3Z)-3-[2-(trifluoromethyl)thioxanthen-9-ylidene]propyl]piperazin-1-yl]ethanol

FLUPENTHIXOL; Flupentixol; trans-flupenthixol; trans-Flupentixol; (E)-Flupenthixol; 53772-85-3; beta-Flupenthixol; ...; 2709-56-0;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)