Olanzapine metabolite

Polymorphisms in CYP2D6 are likely the cause of large interindividual differences in CYP2D6 activities (Ozawa et al., 2004); however, it has been reported that olanzapine clearance is not affected by the CYP2D6 genotype (Carrillo et al., 2003; Nozawa et al., 2008). Moreover, FMO3 also has allelic variants (Shimizu et al., 2014; Yamazaki and Shimizu, 2013). Six genetic variants of FMO3 appear at high frequencies in the Japanese population: E158K, C197fsX, R205C, V257M, E308G, and R500X (Shimizu et al., 2014). Homozygous carriers of FMO3 (p.E308G) in Caucasians have reportedly shown significantly lower plasma concentrations of olanzapine N-oxide than those with wild or heterozygote genotypes (Soderberg et al., 2013). Another group has reported that the frequencies of side effects of olanzapine are associated with FMO3 variants E158K, V257M, and E308G in European Americans, Latin Americans, African Americans, and Asian Americans (Cashman et al., 2008). However, the effects of FMO3 genotypes on olanzapine clearance have not been clarified in a Japanese population.
In the present study, the contributions made by CYP1A2, CYP2D6, and FMO3 to the metabolic clearance of olanzapine were investigated using human liver microsomes, recombinant enzymes, and plasma samples taken from Japanese patients treated with olanzapine. We report that CYP2D6 and FMO3 genotypes and smoking behavior did not affect the olanzapine clearance as a single determinant factor under the present conditions [1].

Microsomal protein concentrations were estimated using a bicinchoninic acid protein assay kit. Activities for N-demethylation, N-oxygenation, and 2-hydroxylation of olanzapine and the rate of substrate disappearance were assayed using high-performance liquid chromatography (HPLC). A typical incubation mixture consisted of 100 mM potassium phosphate buffer (pH 7.4 or 8.4), 5–400 μM olanzapine, human liver microsomes (0.30 mg protein/mL) or recombinant P450s/FMO3 (20 pmol/mL), and an NADPH-generating system (0.25 mM NADP+, 2.5 mM glucose 6-phosphate, and 0.25 unit/mL glucose phosphate dehydrogenase) in a final volume of 0.25 mL, unless otherwise noted, in the absence or presence of the same concentrations of P450 inhibitors as the substrate. To investigate the contribution of FMO3 activity to olanzapine oxygenation, human liver microsomes were preheated at 45 °C for 5 min in the absence of the NADPH-generating system to inactivate FMO3. Incubations were carried out at 37 °C for 45 min and were terminated by adding 250 μL cold acetonitrile. The linearity of olanzapine depletion was confirmed in these incubations. After vortex mixing and centrifugation, the supernatants were injected into an HPLC system. LC analysis was performed using an analytical column (Kinetex XB-C18, 4.6 mm × 100 mm, 2.6 µm; Phenomenex, Torrance, CA, USA) with a guard column (SecurityGuard Ultra C18, 4.6 mm; Phenomenex). The mobile phase was 55% methanol (v/v) containing 75 mM potassium phosphate buffer (pH 7.0), and the flow rate was 0.8 mL/min at 40 °C. The ultraviolet detector was set at 238 nm.
Activities for the N-deethylation of phenacetin, the O-demethylation and N-demethylation of dextromethorphan, and the N-oxygenation of benzydamine were assayed according to previously described methods (Taniguchi-Takizawa et al., 2015; Uehara et al., 2015; Uno et al., 2010). Briefly, standard incubation mixtures consisted of 100 mM potassium phosphate buffer (pH 7.4–8.4), the NADPH-generating system, a substrate (50 μM phenacetin, 400 μM dextromethorphan, or 50 μM benzydamine), and liver microsomes (0.125–0.300 mg protein/mL) in a final volume of 0.20–0.25 mL. Dextromethorphan was incubated with liver microsomes at 37 °C for 15 min, and the reaction was terminated by the addition of 10 μL 60% perchloric acid (w/v). Benzyd amine was incubated at 37 °C for 10 min, and the reaction was terminated by the addition of 200 μL cold acetonitrile. Phenacetin was incubated at 37 °C for 10 min, and the reaction was terminated by the addition of 1.5 mL ethyl acetate and 25 μL 3 M sodium chloride. After extraction, the organic phase was evaporated under a nitrogen stream. Product formation was determined by HPLC with an analytical octadecylsilane (C18) column (4.6 mm × 150 mm, 5 µm), according to the described methods [1].

Metabolism / Metabolites
Olanzapine N-Oxide is a known human metabolite of olanzapine.

Objective: The antipsychotic olanzapine is reportedly metabolized by inducible human cytochrome P450 (CYP) 1A2 and variable copy-number CYP2D6 and polymorphic flavin-containing monooxygenase 3 (FMO3) in different pathways. We investigated individual differences in the metabolite formation and clearance of olanzapine in vitro and in vivo. Methods: Human liver microsomal olanzapine oxidation activities were evaluated, and plasma concentrations of olanzapine were determined in 21 Japanese patients (mean age: 50 years, range: 32-69 years, 14 male and 7 female, including 6 smokers) genotyped for CYP2D6 (*1, *5, and *10) and FMO3 (E158K, C197fsX, R205C, V257M, E308G, and R500X). Results: Furafylline (a CYP1A2 inhibitor), quinidine (a CYP2D6 inhibitor), and heat treatment (inactivates FMO3) suppressed liver microsomal metabolic clearance of olanzapine by approximately 30%. Olanzapine N-demethylation and N-oxygenation were found to be catalyzed by CYP1A2 and CYP2D6 and by CYP2D6 and FMO3, respectively, in experiments using liver microsomes and recombinant enzymes. Plasma concentrations and clearance of olanzapine were not affected by CYP2D6 or FMO3 genotypes or smoking behavior. Conclusions: Olanzapine clearance was not affected by CYP2D6 or FMO3 genotypes or smoking behavior as a single factor under the present conditions because olanzapine clearance is mediated by multiple enzymes involved in two major and one minor pathways.[1]

C17H20N4OS

328.4319

328.135

C, 62.17; H, 6.14; N, 17.06; O, 4.87; S, 9.76

174794-02-6

135409492

Light brown to yellow solid powder

0.58

1

4

1

23

477

0

C[N+]1(CCN(C2NC3=CC=CC=C3N=C3SC(=CC=23)C)CC1)[O-]

LJVNYCDXBXGQIK-UHFFFAOYSA-N

InChI=1S/C17H20N4OS/c1-12-11-13-16(20-7-9-21(2,22)10-8-20)18-14-5-3-4-6-15(14)19-17(13)23-12/h3-6,11,19H,7-10H2,1-2H3

2-methyl-4-(4-methyl-4-oxidopiperazin-4-ium-1-yl)-10H-thieno[2,3-b][1,5]benzodiazepine

174794-02-6; Olanzapine-N-oxide; Olanzapine specified impurity D [EP]; O055W43VHD; LY-170238; 10H-Thieno[2,3-b][1,5]benzodiazepine, 2-methyl-4-(4-methyl-4-oxido-1-piperazinyl)-; 1-Methyl-4-(2-methyl-10H-thieno(2,3-b)(1,5)benzodiazepin-4-yl)piperazin-1-oxide; 10H-Thieno(2,3-b)(1,5)benzodiazepine, 2-methyl-4-(4-methyl-4-oxido-1-piperazinyl)-;

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.)