The metabolic activities of six psychotropic drugs, diazepam, clotiazepam, tofisopam, etizolam, tandospirone, and imipramine, were determined for 14 isoforms of recombinant human hepatic cytochrome P450s (CYPs) and human liver microsomes by measuring the disappearance rate of parent compounds. In vitro kinetic studies revealed that Vmax/Km values in human liver microsomes were the highest for tofisopam, followed by tandospirone>clotiazepam>imipramine, diazepam, and etizolam. Among the recombinant CYPs, CYP3A4 exhibited the highest metabolic activities of all compounds except for clotiazepam and imipramine. The metabolism of clotiazepam was catalyzed by CYP2B6, CYP3A4, CYP2C18, and CYP2C19, and imipramine was metabolized by CYP2D6 most efficiently. In addition, the metabolic activities of diazepam, clotiazepam, and etizolam in human liver microsomes were inhibited by 2.5 microM ketoconazole, a CYP3A4 inhibitor, by 97.5%, 65.1%, and 83.5%, respectively, and the imipramine metabolism was not detected after the addition of 1 or 10 microM quinidine, a CYP2D6 inhibitor. These results suggest that the psychotropic drugs investigated are metabolized predominantly by CYP3A4, except that CYP2D6 catalyzes the metabolism of imipramine. In addition, this approach based on the disappearance rate appears to be useful for the identification of the responsible CYP isoform(s) of older drugs, for which metabolic profiles have not been reported.[1]

Absorption, Distribution and Excretion
Etizolam is well absorbed from the intestines with a biological bioavailability of 93% following oral administration. After a single oral dosing of 0.5mg etizolam, it takes approximately 0.9 hours to reach the peak plasma concentration of 8.3 ng/mL.
In a rat study, the amounts of etizolam excreted was 30% in urine was 70% in feces, while the values in a mouse study were 40% in urine and 60% in feces.
Apparent distribution volume was 0.9 ± 0.2 L/kg following a single oral doing of 0.5mg etizolam.

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Metabolism / Metabolites
Biotransformation of etizolam is extensive and involves hydroxylation and conjugation. The main metabolite formed via 1'-hydroxylation is α-hydroxyetizolam which retains pharmacological activity comparable to that of the parent drug, indicating that the action of metabolites may contribute to the clinical effects of etizolam. CYP3A4 is predicted to be the main CYP enzyme responsible for mediating etizolam metabolism. CYP2C18 and CYP2C19 are also involved in the metabolic pathways.
Etizolam has known human metabolites that include 7-(2-chlorophenyl)-4-ethyl-13-methyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-ol and alpha-Hydroxyetizolam.

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Biological Half-Life
The average elimination half life of etizolam following a single oral dose of 0.5mg is 3.4 hours but may be increased up to 17 hours depending on the rate of metabolism. The main metabolite α-hydroxyetizolam displays a longer elimination half life of 8.2 hours.

Toxicity Summary
Benzodiazepines bind nonspecifically to benzodiazepine receptors BNZ1, which mediates sleep, and BNZ2, which affects affects muscle relaxation, anticonvulsant activity, motor coordination, and memory. As benzodiazepine receptors are thought to be coupled to gamma-aminobutyric acid-A (GABAA) receptors, this enhances the effects of GABA by increasing GABA affinity for the GABA receptor. Binding of the inhibitory neurotransmitter GABA to the site opens the chloride channel, resulting in a hyperpolarized cell membrane that prevents further excitation of the cell.

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Etizolam is not approved for marketing in the United States by the U.S. Food and Drug Administration. Very little information is available on the passage of etizolam into milk. An alternate drug is preferred, especially while nursing a newborn or preterm infant. If etizolam is used, monitor the infant for sedation, poor feeding and poor weight gain.
◉ Effects in Breastfed Infants
A woman took etizolam 1 mg and trazodone 50 mg once daily for 3 months postpartum. Her infant was over 50% breastfed and demonstrated no adverse reactions at the 1- and 3-month checkups.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

Biol Pharm Bull.2005 Sep;28(9):1711-6;Curr Drug Metab.2008 Oct;9(8):827-44.

Etizolam is an organic molecular entity.
4-(2-chlorophenyl)-2-ethyl-9-methyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine, its salts, isomers, and salts of isomers is a DEA Schedule I controlled substance. Substances in the DEA Schedule I have no currently accepted medical use in the United States, a lack of accepted safety for use under medical supervision, and a high potential for abuse. It is a Temporary listing of substances subject to emergency scheduling substance.
Etizolam is a thienodiazepine which is chemically related to benzodiazepine (BDZ) drug class; it differs from BDZs in having a benzene ring replaced with a thiophene ring. It is an agonist at GABA-A receptors and possesses amnesic, anxiolytic, anticonvulsant, hypnotic, sedative and skeletal muscle relaxant properties. Initially introduced in 1983 in Japan as treatment for neurological conditions such as anxiety and sleep disorders, etizolam is marketed in Japan, Italy and India. It is not approved for use by FDA in the US; however it remains unscheduled in several states and is legal for research purposes.
According to the Italian P.I. sheet etizolam belongs to a new class of diazepines, thienotriazolodiazepines. This new class is easily oxidized, rapidly metabolized, and has a lower risk of accumulation, even after prolonged treatment. Etizolam has an anxiolytic action about 6 times greater than that of diazepam. Etizolam produces, especially at higher dosages, a reduction in time taken to fall asleep, an increase in total sleep time and a reduction in the number of awakenings. During tests there were not substantial changes in deep sleep. There is a reduction of REM sleep. In EEG tests of healthy volunteers Etizolam showed some characteristics of tricyclic antidepressants. Etizolam (marketed under the brand name Etilaam, Etizola, Sedekopan, Pasaden or Depas) is a thienodiazepine drug which is a benzodiazepine analog. The etizolam molecule differs from a benzodiazepine in that the benzene ring has been replaced by a thiophene ring. It possesses amnesic, anxiolytic, anticonvulsant, hypnotic, sedative and skeletal muscle relaxant properties.

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Drug Indication
Indicated for the treatment of generalized anxiety disorder with depression, panic disorder and insomnia.

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Mechanism of Action
Etizolam is selectively a full agonist at GABA-A receptors to increase GABAergic transmission and enhance GABA-induced Cl- currents. It is reported to bind to the benzodiazepine binding site which is located across the interface between the alpha and gamma subunits. Benzodiazapines are reported to only bind to receptors that contain gamma 2 and alpha 1/2/3/5 subunits. Alpha-1-containing receptors mediate the sedative effects of etizolam whereas alpha-2 and alpha-3 subunit-containing receptors mediate the anxiolytic effect. Etizolam shows high potency and affinity towards GABA-A receptor with alpha 1 beta 2 gamma 2S subunit combination. By binding to the regulatory site of the receptor, etizolam potentiates GABA transmission by facilitating the opening of GABA-induced chloride channels. Etizolam is a specific antagonist at PAFR. It inhibits PAF-induced platelet aggregation by inhibiting PAF binding to the receptors located on the surface of platelets with an IC50 of 22nM.

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Pharmacodynamics
Etizolam is a CNS depressant with anxiolytic, anticonvulsant, sedative-hypnotic and muscle relaxant effects. It acts on the benzodiazepine site of the GABA-A receptor as an agonist to increase inhibitory GABAergic transmission throughout the central nervous system. Studies indicate that etizolam mediates its pharmacological actions with 6 to 10 times more potency than that of diazepam. Clinical human studies performed in Italy showed clinical effectiveness of etizolam in relieving symptoms in patients with generalized anxiety disorders with depressive symptoms. Etizolam also mediates imipramine-like neuropharmacological and behavioral effects, as well as minor effects on cognitive functioning. It is shown to substitute the actions of a short-acting barbiturate, pentobarbitol, in a drug discrimination study. Etizolam is an antagonist at platelet-activating-factor (PAF) receptor and attenuates the recurrence of chronic subdural hematoma after neurosurgery in clinical studies. It is shown to inhibit PAF-induced bronchoconstriction and hypotension.

C17H15CLN4S

342.07

40054-69-1

3307

Typically exists as solid at room temperature

147-148

2.6

0

4

2

23

474

0

VMZUTJCNQWMAGF-UHFFFAOYSA-N

InChI=1S/C17H15ClN4S/c1-3-11-8-13-16(12-6-4-5-7-14(12)18)19-9-15-21-20-10(2)22(15)17(13)23-11/h4-8H,3,9H2,1-2H3

7-(2-Chlorophenyl)-4-ethyl-13-methyl-3-thia-1,8,11,12-tetraazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaene

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.)