Midazolam metabolite
Gamma-aminobutyric acid type A (GABAA) receptor (indirect). 1'-Hydroxymidazolam is the principal active metabolite of midazolam. Like the parent drug, it acts as a positive allosteric modulator of the GABAA receptor, binding to the benzodiazepine site. This binding enhances the affinity of the receptor for GABA, leading to increased chloride ion channel opening frequency, neuronal hyperpolarization, and central nervous system depression. It contributes additively to the overall sedative, anxiolytic, anticonvulsant, and muscle-relaxant effects of midazolam.

In mouse wild type cultures, neocortical slices treated with 1'-hydroxymidazolam (5 nM-1 µM) exhibit a consistent decrease in network activity at concentrations up to 1 µM and a significant inhibition of action potential firing rates at concentrations of 100 nM or higher [2].

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In cell-free systems, 1'-Hydroxymidazolam is not directly tested for receptor binding activity. Instead, it is used as a substrate to study CYP3A4/5 enzyme kinetics. The compound is formed by incubating midazolam with human liver microsomes or recombinant CYP3A4 enzyme in the presence of NADPH. Its production is quantified by LC-MS/MS to determine enzyme activity. The conversion of midazolam to 1'-Hydroxymidazolam is the most commonly used probe reaction for in vitro CYP3A4/5 phenotyping. The Michaelis-Menten constant (Km) for this reaction is typically 1-3 uM.

The main purpose of this study is to demonstrate the possibility of increase in the systemic exposure of drug metabolites by CYP-inhibition or acute hepatitis. Midazolam (MDZ) was used as a model substrate of CYP3A and 1-aminobenzotriazole (ABT) was used as a CYP-inhibitor. After oral pretreatment with ABT, MDZ was intravenously injected to rats and the plasma profiles of MDZ and its primary metabolites, 1'-hydroxy MDZ and 4-hydroxy MDZ, were observed. In the ABT-pretreatment rats, plasma AUCs of both metabolites were much larger than those in control rats, demonstrating a higher systemic exposure of metabolites under CYP-inhibited condition. Furthermore, kinetic analysis revealed that the amount of both metabolites entered into the systemic circulation increased significantly (about 5-times). Increases in the systemic exposure of the primary metabolites of MDZ were also observed in the acute hepatitis rats induced by CCl4-pretreatment. As underlying mechanisms, it was speculated that ABT inhibited the subsequent metabolism of primary metabolites of MDZ in the hepatocytes and enhanced their release to the systemic circulation. In vitro study with rat liver microsomes supported this speculation. In conclusion, this study showed the complexity of PK profiles of drug metabolites, which might lead to new aspects on their safety issue[1].

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In vitro, 1'-Hydroxymidazolam exhibits pharmacological activity at the GABAA receptor, though it is generally considered to be equipotent or slightly less potent than midazolam. Its cellular effects include inhibiting neuronal activity by potentiating GABAergic inhibition. In transfected HEK293 cells expressing different GABAA receptor subunits, it enhances GABA-induced currents. The metabolite has a longer elimination half-life than midazolam in some species, leading to a potential accumulation of active metabolite during prolonged infusions or in patients with hepatic impairment. It is also used as a standard in assays for CYP3A4 activity.

To evaluate 1'-Hydroxymidazolam as a product of CYP3A4 metabolism, a standard in vitro enzyme assay is performed. Recombinant human CYP3A4 is incubated with midazolam (1-10 uM), an NADPH-regenerating system, and varying concentrations of a test compound (if testing inhibition) in a phosphate buffer (pH 7.4). The reaction is started by adding NADPH and incubated at 37degC for 5-30 minutes (ensuring linear product formation). The reaction is stopped by cooling on ice and adding acetonitrile. The amount of 1'-Hydroxymidazolam formed is quantified by LC-MS/MS or HPLC-UV. Kinetic parameters (Km, Vmax) are calculated from the Michaelis-Menten equation.

1'-Hydroxymidazolam itself is not typically used as a drug; rather, it is a metabolite measured in cell-based systems to assess CYP3A4 activity. Hepatocytes (primary human or cryopreserved) are cultured in 24-well plates and incubated with midazolam (1-10 uM) for 0.5-4 hours at 37degC. Aliquots of the culture medium are collected at various time points, and the concentration of 1'-Hydroxymidazolam is measured by LC-MS/MS. The rate of metabolite formation is used as a direct measure of the intrinsic metabolic clearance of the hepatocytes. This assay is a gold standard for predicting in vivo drug-drug interactions involving CYP3A4.

The benzodiazepine midazolam is widely used in critical care medicine. Midazolam has a clinically active metabolite, 1-hydroxymidazolam. The contribution of 1-hydroxymidazolam to the effects of midazolam is controversial. The aim of the current study was to compare the actions of midazolam and 1-hydroxymidazolam on network activity of cortical neurons. Midazolam depressed neuronal activity at a low concentration of 5 nM. When midazolam concentration was increased, it depressed neuronal discharge rates in a biphasic manner. In comparison, 1-hydroxymidazolam did not depress the cortical network activity at low nanomolar concentrations. Higher concentrations of 1-hydroxymidazolam consistently inhibited neuronal activity. Moreover, midazolam shortened cortical up states at low, but not at high concentrations, while the opposite effect was observed with 1-hydroxymidazolam. The network depressant action of midazolam at low concentrations was absent in slices from GABAA receptor α1(H101R)mutant mice. The α1(H101R)mutation renders α1-subunit containing GABAA receptors insensitive towards benzodiazepines. This GABAA receptor subtype is thought to mediate sedation. As midazolam is more potent than its metabolite 1-hydroxymidazolam, the major clinical effects are thus likely caused by midazolam itself. However, 1-hydroxymidazolam could add to the effects of midazolam, especially after the application of high doses of midazolam, and in case of impaired drug metabolism.[2]

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In vivo, the formation of 1'-Hydroxymidazolam is used as a biomarker for CYP3A4 activity in preclinical species and humans. For a typical in vivo PK study, midazolam (e.g., 1 mg/kg IV or 2 mg/kg PO) is administered to rats, dogs, or monkeys. Serial blood samples are collected over a 24-hour period, and plasma concentrations of both midazolam and its metabolite 1'-Hydroxymidazolam are quantified by LC-MS/MS. The metabolic ratio (AUC_metabolite / AUC_parent) is calculated to assess the extent of first-pass metabolism and the activity of CYP3A. In drug-drug interaction studies, changes in the plasma levels of 1'-Hydroxymidazolam indicate whether a test compound inhibits or induces CYP3A4.

Metabolism / Metabolites
1'-Hydroxymidazolam is a known metabolite of midazolam in the human body.

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The PK properties of 1'-Hydroxymidazolam are derived from midazolam administration. It is rapidly formed after oral or intravenous administration of midazolam, with a Tmax of 0.5-1 hour. The metabolite has a terminal elimination half-life of approximately 1.5-4 hours, which is slightly longer than the parent drug (2-3 hours). It is more polar than midazolam and is primarily eliminated via the kidneys after conjugation (glucuronidation). Its AUC can exceed that of the parent drug, especially after oral administration due to first-pass metabolism. 1'-Hydroxymidazolam is also a substrate for CYP3A4 and can be further metabolized to 1'-Hydroxymidazolam glucuronide or to 4-hydroxymidazolam.

Metabolism/Metabolites
1'-Hydroxymidazolam is a known metabolite of midazolam in the human body.

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The toxicity of 1'-Hydroxymidazolam is primarily related to its pharmacological activity as a GABAA receptor agonist. Adverse effects are similar to those of benzodiazepines and include sedation, respiratory depression, ataxia, and amnesia. In overdose, it can cause pronounced CNS depression and coma. It is not associated with specific organ toxicity; however, because it is a metabolite, its accumulation in patients with hepatic or renal impairment can lead to prolonged sedative effects. In research settings, it is used as a reference standard and is not administered to humans as a pure compound.

[1]. Increase in the systemic exposure of primary metabolites of Midazolam in rat arising from CYP inhibition or hepatic dysfunction. Drug Metab Pharmacokinet. 2017 Feb; 32(1): 69-76.

[2]. Differential depression of neuronal network activity by midazolam and its main metabolite 1-hydroxymidazolam in cultured neocortical slices. Sci Rep. 2017 Jun 14; 7(1): 3503.

1-Hydroxymidazolam is a midazolam derivative in which a hydrogen atom on the methyl group is replaced by a hydroxyl group. It is the major metabolite of the anesthetic midazolam. It is a drug metabolite as well as a metabolite in human serum and urine. It belongs to the midazolam class of compounds, organochlorine compounds, monofluorobenzenes, and aromatic primary alcohols. Its function is related to that of midazolam.

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1'-Hydroxymidazolam (CAS 59468-90-5) is a key active metabolite of midazolam, used extensively as a diagnostic probe for CYP3A4/5 enzyme activity and in drug interaction studies. It is also employed as a reference standard in bioanalytical method development for the quantification of midazolam and its metabolites. The compound is not approved as a standalone drug but is a crucial research tool in drug metabolism and pharmacokinetics (DMPK). It is typically provided as a neat powder or in solution for use as a standard in LC-MS/MS assays. It should be stored at -20degC.

C18H13N3OFCL

341.76672

341.073

59468-90-5

1'-Hydroxymidazolam-d4;1781843-10-4;1'-Hydroxymidazolam-13C3;1189677-14-2;1'-Hydroxymidazolam-13C6;1261396-36-4

107917

White to off-white solid powder

2.7

1

4

2

24

491

0

C1=CC=C(C(=C1)C2=NCC3=CN=C(CO)N3C4=C2C=C(C=C4)Cl)F

QHSMEGADRFZVNE-UHFFFAOYSA-N

InChI=1S/C18H13ClFN3O/c19-11-5-6-16-14(7-11)18(13-3-1-2-4-15(13)20)22-9-12-8-21-17(10-24)23(12)16/h1-8,24H,9-10H2

[8-chloro-6-(2-fluorophenyl)-4H-imidazo[1,5-a][1,4]benzodiazepin-1-yl]methanol

1'-hydroxymidazolam; 1-Hydroxymidazolam; 59468-90-5; 1'-Hydroxy Midazolam; alpha-Hydroxymidazolam; 1-Hydroxymethylmidazolam; 1-OH-Mdz; ALPHA-HYDROXY-MIDAZOLAM;

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.

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.)