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
Following oral administration, flupentixol is readily absorbed from the gastrointestinal tract, with oral bioavailability of about 40%. Tmax ranges from three to eight hours. Steady-state plasma levels are achieved in about seven days and following once-daily oral administration of 5 mg flupentixol, the mean minimum steady-state level was about 1.7 ng/mL (3.9 nmol/L). From the site of intramuscular injection, esterified flupentixol diffuses slowly from the oil solution and is slowly released into the extracellular fluid and the circulation to be distributed to different tissues. Peak drug concentrations are reached between four and seven days following intramuscular injection. Intramuscularly administered flupentixol is detectable in the blood three weeks after injection and reaches steady-state concentrations after about three months of repeated administration.
Fecal excretion is more predominant than renal excretion. In the feces, flupentixol is recovered in the feces mainly as the unchanged form, as well as its lipophilic metabolites, such as dealkyl-flupentixol. Flupentixol is recovered in the urine as the unchanged form as well as its hydrophilic sulfoxide and glucuronide metabolites.
The apparent volume of distribution is about 14.1 L/kg. Following administration, the highest levels of flupentixol are found in the lungs, liver, and spleen. Lower concentrations of the drug are found in the blood and brain.
Following oral administration, the mean systemic clearance is about 0.29 L/min.

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Metabolism / Metabolites
Flupentixol is metabolized in the liver via sulfoxidation, dealkylation, and glucuronidation to form pharmacologically inactive metabolites. Flupentixol decanoate, the active ingredient in the intramuscular formulation, is hydrolyzed to flupentixol.

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Biological Half-Life
The elimination half-life is about 35 hours following oral administration and three weeks following intramuscular administration.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Flupenthixol is not approved for marketing in the United States by the U.S. Food and Drug Administration, but is available in other countries. Limited information indicates that maternal oral doses of up to 4 mg daily or depot injections of 40 mg every 2 weeks produced low levels in milk and breastfed infants' serum, and caused no adverse developmental consequences. A safety scoring system finds flupenthixol possible to use cautiously during breastfeeding. Until more data are available, flupenthixol should be used with careful infant monitoring during breastfeeding.
◉ Effects in Breastfed Infants
A woman took flupenthixol 1 mg and nortriptyline 100 mg daily during pregnancy and flupenthixol 4 mg and nortriptyline 125 mg daily immediately postpartum. She exclusively breastfed her infant. Over a 4-month period, the infant showed no signs of adverse drug effects and had normal motor development with a maternal dosage of flupenthixol 2 mg daily and nortriptyline 75 mg daily.
◉ Effects on Lactation and Breastmilk
Flupenthixol can increase serum prolactin and has caused galactorrhea. The prolactin level in a mother with established lactation may not affect her ability to breastfeed.

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Protein Binding
Flupentixol is 99% bound to plasma proteins.

[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-flupenthixol is a flupenthixol in which the double bond adopts a cis-configuration. It has a role as a dopaminergic antagonist. It is a conjugate base of a cis-flupenthixol(2+).
Flupentixol is an antipsychotic drug of the thioxanthene group. It exists in two geometric isomers, the trans(E) and pharmacologically active cis(Z) forms. Flupentixol decanoate is one of the active ingredients found in injectable drug formulations: it is produced by esterification of cis(Z)‐flupentixol with decanoic acid. Flupentixol is an antagonist of both D1 and D2 dopamine receptors. Available as oral tablets or long-acting intramuscular injections, flupentixol is marketed under brand names such as Depixol and Fluanxol. It is approved for use in Canada and other countries around the world, but not in the US. It is used for the management of chronic schizophrenia in patients whose main manifestations do not include excitement, agitation or hyperactivity. It has been marketed to manage symptoms of depression in patients who may or may not exhibit signs of anxiety. In combination with [melitracen], flupentixol is used to manage symptoms of anxiety, depression, and asthenia.
A thioxanthene neuroleptic that, unlike CHLORPROMAZINE, is claimed to have CNS-activating properties. It is used in the treatment of psychoses although not in excited or manic patients. (From Martindale, The Extra Pharmacopoeia, 30th ed, p595)

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Drug Indication
Flupentixol is indicated for maintenance therapy of chronic schizophrenic patients whose main manifestations do not include excitement, agitation or hyperactivity. It is indicated for the management of depression in adult patients who may, or may not, also be showing signs of anxiety. Flupentixol in combination with [melitracen] is indicated to manage symptoms of anxiety, depression, and asthenia in adults.

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Mechanism of Action
The mechanism of action of flupentixol is not completely understood. The antipsychotic actions are mainly thought to arise from cis(Z)-flupentixol, the active stereoisomer, acting as an antagonist at both dopamine D₁ and D₂ receptors with equal affinities. Schizophrenia is a mental illness characterized by positive (such as hallucinations and delusions) and negative (such as affect flattening and apathy) symptoms. While several neurotransmitter systems are implicated in the pathophysiologic processes leading to the development of symptoms, the dopamine and glutamate systems have been extensively studied. It is generally understood that positive symptoms of schizophrenia arise from a dysregulated striatal dopamine pathway, leading to hyperstimulation of D₂ receptors. Many antipsychotic agents work by blocking D₂ receptors as antagonists; similarly, cis(Z)-flupentixol, the active stereoisomer, is an antagonist at D₂ receptors. However, there is now evidence that antipsychotic agents can work by blocking other dopamine receptor subtypes, such as D₁, D₃, or D₄ receptors. One study showed that cis(Z)-flupentixol is an antagonist at both dopamine D₁ and D₂ receptors with equal affinities, and binds to D3 and D4 receptors with lower affinities. It also binds to alpha-1 adrenoceptors. Antidepressant effects of flupentixol are understood to be mediated by antagonism at 5-HT_2A receptors, which are commonly downregulated following repeated antidepressant treatment. Flupentixol also binds to 5-HT_2C receptors.

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Pharmacodynamics
Flupentixol is an antipsychotic agent with anxiolytic and mild sedative actions. It exerts weak anticholinergic and adrenergic effects. It possesses antiemetic actions. As flupentixol works by antagonizing dopamine actions, it can cause extrapyramidal effects, mostly at doses greater than 10 mg. In clinical trials, flupentixol-induced extrapyramidal effects have been managed with anti-Parkinsonian drugs. Drug esterification in the intramuscular formulation of the drug results in slow release of the drug from the injection site and a prolonged duration of action. Flupentixol has been investigated for use in mild to moderate depression: compared to other antidepressant agents, flupentixol has a rapid onset of action, where antidepressive effects were observed within the first two to three days after administration. As with other antipsychotic agents, flupentixol can cause QTc prolongation and increase the risk of arrhythmias. In clinical trials, flupentixol was associated with the risk of cardiovascular disease, cerebrovascular adverse events, stroke, and venous thromboembolism. Flupentixol can elevate the levels of prolactin; however, the clinical significance of hyperprolactinemia caused by neuroleptic drugs is unclear. Long-term hyperprolactinemia, when associated with hypogonadism, may lead to decreased bone mineral density in both female and male subjects. Interestingly, recent studies show that flupentixol exhibits anti-tumour properties alone or synergistically with other anticancer drugs like gefitinib. One study demonstrated that _in vitro_, flupentixol docks to the ATP binding pocket of phosphatidylinositol 3-kinase (PI3K), a lipid kinase that activates signalling pathways that are often hyperactivated in some cancers. Flupentixol inhibited the PI3K/AKT pathway and survival of lung cancer cells _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.)