vilazodone metabolite
Vilazodone (parent drug) is a selective serotonin reuptake inhibitor (SSRI) and a partial agonist of the 5-HT1A receptor. The carboxylic acid metabolite has significantly reduced (or negligible) binding affinity for the serotonin transporter (SERT) and 5-HT1A receptors compared to the parent drug, and is considered pharmacologically inactive at therapeutic concentrations.

The semi-quantitative estimation of VLZ (Vilazodone) and metabolites was carried out on the basis of percent (%) count. The relative percent abundance for Vilazodone (VLZ) and each metabolite (M1-M12) formed in urine, faeces and plasma are summarized in Fig. 2 (a-c). As the metabolism studies were conducted without any reference standards, there may be uncertainty regarding the estimation of metabolites and therefore ion counts have been utilized to carry out semiquantitative estimation of metabolites.[32, 33] Based on the present study, it was observed that VLZ was found in all in vivo samples and comparatively higher percent in faeces (13.93%). This article is protected by copyright. All rights reserved. In urine, VLZ was extensively metabolized which led to the formation of nine metabolites. Metabolites M4 (10.86%) and M11 (27.02%) were the major metabolites observed in urine whereas other metabolites were detected in minor amounts. Metabolites M6 (47.7%) and M2 (11.69%) were formed at higher levels in faeces including VLZ, while other metabolites were minor. Three metabolites were detected in plasma, among which metabolite M8 (3.54%) was the only metabolite formed in higher level and other metabolites (M4 and M11) were detected in minor amounts. Minor amount of M4 and M8 metabolites were detected in both HLM and RLM.[1]

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Vilazodone inhibits the serotonin transporter (SERT) with an IC50 of approximately 0.2-0.5 nM and is a 5-HT1A partial agonist with an EC50 of approximately 2 nM. The carboxylic acid metabolite has been shown to have little to no measurable activity in these assays, implying that it does not contribute to the antidepressant efficacy of vilazodone.

Vilazodone is a selective serotonin reuptake inhibitor (SSRI) used for the treatment of major depressive disorder (MDD). An extensive literature search found few reports on the in vivo and in vitro metabolism of vilazodone. Therefore, we report a comprehensive in vivo and in vitro metabolic identification and structural characterization of vilazodone using ultrahigh-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF/MS/MS) and in silico toxicity study of the metabolites.[1]
Methods: To identify in vivo metabolites of vilazodone, blood, urine and faeces samples were collected at different time intervals starting from 0 h to 48 h after oral administration of vilazodone to Sprague-Dawley rats. The in vitro metabolism study was conducted with human liver microsomes (HLM) and rat liver microsomes (RLM). The samples were prepared using an optimized sample preparation approach involving protein precipitation followed by solid-phase extraction. The metabolites have been identified and characterized by using LC/ESI-MS/MS.[1]
Results: A total of 12 metabolites (M1-M12) were identified in in vivo and in vitro matrices and characterized by LC/ESI-MS/MS. The majority of the metabolites were observed in urine, while a few metabolites were present in faeces and plasma. Two metabolites were observed in the in vitro study. A semi-quantitative study based on percentage counts shows that metabolites M11, M6 and M8 were observed in higher amounts in urine, faeces and plasma, respectively.[1]
Conclusions: The structures of all the 12 metabolites were elucidated by using LC/ESI-MS/MS. The study suggests that vilazodone was metabolized via hydroxylation, dihydroxylation, glucuronidation, oxidative deamination, dealkylation, dehydrogenation and dioxidation. All the metabolites were screened for toxicity using an in silico tool.[1]

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Vilazodone (2-20 mg/kg orally) in rat models of depression (e.g., FST) significantly reduces immobility time. The carboxylic acid metabolite, however, is not known to contribute to these antidepressant-like effects. Following oral administration, vilazodone is rapidly absorbed and metabolized to the carboxylic acid metabolite (M10). In a rat PK study, the Cmax of the metabolite was ~26 ng/mL.

In vitro metabolite generation[1]
The in vitro metabolite generation studies were performed according to reported methodology. To conduct in vitro metabolism study, 25 µl of HLM (Human Liver Microsomes) and RLM (Rat Liver Microsomes) suspensions (20 mg ml-1 ) were added to two different eppendorf tubes containing 420 µl of 100 mM phosphate buffer (pH 7.4) respectively. Microsomes were preconditioned for 5 min at 37 °C. 5 µl of 1mM stock solution of the drug was added to each tube. The metabolic reaction was initiated by addition of 50 µl of 10 mM NADPH (cofactor). Control samples consisted of phosphate buffer instead of cofactor. Initially an aliquot of 100 µl was withdrawn as zero time point sample and was quenched instantly with equal volume of chilled ACN, succeeded by vortexing. The second aliquot (400 µl) was incubated in the incubator shaker with continuous shaking (50 rpm) at 37 °C for the duration of 2 h, and later processed in similar fashion. All the samples were centrifuged at 10000 rpm for 10 min and the supernatants were subjected to solid phase extraction (SPE) for enrichment of metabolites and removal of interference.

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Not applicable (the metabolite has no specific pharmacological activity and thus does not undergo dedicated enzyme/receptor binding assays; it is used as an analytical standard). If binding assays were performed, the protocol would mirror that of the parent drug: membrane preparations from HEK-293 cells expressing human SERT are incubated with [3H]citalopram and varying concentrations of the metabolite. However, Vilazodone carboxylic acid is used primarily for quantification, not activity assessment.

Not applicable (the metabolite has no direct biological activity, and thus is not tested in cellular activity assays). For standard antidepressant cellular assays, rat brain synaptosomes or transfected HEK-293 cells expressing SERT are pre-incubated with vilazodone, but this is not applicable to the carboxylic acid metabolite. Instead, the metabolite is used in cytotoxicity assays: HepG2 or primary hepatocytes are treated with the metabolite (0.1-100 uM) for 24 h to assess safety (MTT viability).

For in vivo metabolic profiling of VLZ (Vilazodone), male Sprague–Dawley rats (200-220 gm) were used.[1]
In vivo Sample preparation Vilazodone (VLZ) was orally administered to rats in the form of 0.5% carboxymethyl-cellulose suspension at a dose of 20 mg kg-1 . Animals were given access to the food after 4 h of the drug administration. Blood samples (0.2 mL) were collected from rats (n=6) in 0.5 mL eppendorf tubes through retro-orbital route according to known pharmacokinetics. The plasma was separated from the blood samples by centrifugation at 6000 rpm for 10 min at 4 °C and stored at -80 °C until analysis. Protein precipitation was done by addition of three volumes of ACN to urine and plasma followed by vortexing for 2-3 min and centrifugation at 10000 rpm for 10 min. The supernatant was concentrated via nitrogen evaporation and the remaining aqueous portion was subjected to SPE using strata C18-E cartridges. The eluted portion was submitted for analysis. The faeces and urine This article is protected by copyright. All rights reserved. samples were collected before drug administration and at intervals of 0-8, 8-24, 24-48 h post dose from another set of animals (n= 6). Sample aliquots were pooled together and stored at - 80 °C until analysis. In case of faeces, equal volumes of water and ACN were added. The mixture was vortexed to slurry which was subjected to centrifugation at 10000 rpm for 10 min and treated in a similar way like urine and plasma samples.[1]

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Not applicable (the metabolite is not administered alone in efficacy studies; it is a byproduct of parent drug metabolism). In male Sprague-Dawley rats (n=4-6), vilazodone is administered orally at 4 mg/kg. Blood samples are collected at 0.5, 1, 2, 4, 6, 8, and 24 h post-dose. Plasma is analyzed by LC-MS/MS for vilazodone and its carboxylic acid metabolite (M10) using appropriate internal standards. Pharmacokinetic parameters (Cmax, Tmax, AUC, t½) for both the parent and metabolite are determined.

Vilazodone is well absorbed (oral bioavailability ~70-80%) and has a terminal half-life of ~24-28 h in humans. It is ~94-96% protein bound. The carboxylic acid metabolite (M10) is formed via esterase-mediated hydrolysis of vilazodone and possibly via amidase pathways. The metabolite is primarily eliminated in urine, with minimal circulating levels relative to the parent drug.

The carboxylic acid metabolite is an analytical standard, not a therapeutic agent, and thus human toxicity data are not relevant. The parent drug vilazodone is generally well tolerated. Common AEs of vilazodone: diarrhea (28%, highest among SSRIs), nausea (23%), insomnia, and headache. The carboxylic acid metabolite is not known to contribute to these side effects.

[1]. Identification and characterization of vilazodone metabolites in rats and microsomes by ultrahigh-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry. Rapid Communications in Mass Spectrometry. 2017. 31(23), 1974–1984.

This study used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify and characterize 12 VLZ metabolites in biological matrices (rat urine, feces, plasma, human liver microsomes, and rat liver microsomes). Sample pretreatment involved protein precipitation and solid-phase extraction. The metabolites (M1-M12) were systematically characterized by comparing the mass spectra of VLZ and the metabolites and by precise mass determination. In vitro experiments showed that VLZ can generate hydroxylated and dihydroxylated metabolites. The in vivo biotransformation pathways of VLZ include hydroxylation, glucuronidation, dehydrogenation, and oxidative deamination. This study used TOPKAT software to perform computer simulation screening of the metabolites. This study provides comprehensive information on the in vivo and in vitro metabolism of VLZ in rats. The structural information of the metabolites is helpful for developing new molecules with better therapeutic effects. [1]

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Vilazodone HCl (Viibryd) was FDA-approved in 2011 for the treatment of major depressive disorder (MDD). The carboxylic acid metabolite is a research standard used for the identification and quantification of vilazodone and its metabolites in biological fluids, supporting pharmacokinetic (PK) studies, metabolism pathway elucidation, and forensic or clinical toxicology.

C26H26N4O3

442.5096

442.2

163521-19-5

Vilazodone Hydrochloride;163521-08-2;Vilazodone;163521-12-8

11201569

White to light yellow solid powder

4.631

2

6

7

33

730

0

RSXUEYFLDNUILS-UHFFFAOYSA-N

InChI=1S/C26H26N4O3/c27-16-18-4-6-23-22(13-18)19(17-28-23)3-1-2-8-29-9-11-30(12-10-29)21-5-7-24-20(14-21)15-25(33-24)26(31)32/h4-7,13-15,17,28H,1-3,8-12H2,(H,31,32)

5-[4-[4-(5-cyano-1H-indol-3-yl)butyl]piperazin-1-yl]-1-benzofuran-2-carboxylic acid

5-(4-(4-(5-Cyano-1H-indol-3-yl)butyl)piperazin-1-yl)benzofuran-2-carboxylic acid; Vilazodone Carboxylic acid; Vilazodone metabolite M10; 5-[4-[4-(5-cyano-1H-indol-3-yl)butyl]piperazin-1-yl]-1-benzofuran-2-carboxylic acid; 93K783WZV4; 2-Benzofurancarboxylic acid, 5-[4-[4-(5-cyano-1H-indol-3-yl)butyl]-1-piperazinyl]-; 5-[4-[4-(5-cyano-1H-indol-3-yl)butyl]-1-piperazinyl]-2-Benzofurancarboxylic 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)

DMSO : ~4 mg/mL (~9.04 mM)

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.)