CYP2C8 (IC50 = 1.4 μM); CYP2B6 (IC50 = 2.0 μM); CYP3A (IC50 = 2.3 μM); CYP2C19 (IC50 = 2.5 μM); CYP2D6 (IC50 = 2.5 μM); CYP2E1 (IC50 = 2.7 μM); CYP2C9 (IC50 = 3.3 μM); CYP2J2 (IC50 = 3.3 μM); CYP2A6 (IC50 = 3.8 μM (IC50); CYP1A2 (IC50 = 4.0 μM)

With IC50 values of 5.2 μM and 3.0 μM, respectively, DL-Acetylshikonin inhibits the CYP3A-mediated metabolism of testosterone (testosterone) and nifedipine, suggesting that it inhibits CYP3A activity in a substrate-independent way [1]. Time-dependent inhibition is not seen by DL-Acetylshikonin [1].

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Acetylshikonin is a biologically active compound with anti-cancer and anti-inflammatory activity, which is isolated from the roots of Lithospermum erythrorhizoma. An inhibitory effect of acetylshikonin against CYP2J2 activity was discovered recently. Based on this result, this study was expanded to evaluate the inhibitory effects of acetylshikonin against nine different cytochrome P450 (P450) isoforms in human liver microsomes (HLMs) using substrate cocktails incubation assay. Acetylshikonin showed a strong inhibitory effect against all P450s tested with IC50 values of 1.4-4.0 μ m. Pre-incubation of acetylshikonin with HLMs and NADPH did not alter the inhibition potency, indicating that acetylshikonin is not a mechanism-based inhibitor. SKF-525A, a widely used non-specific P450 inhibitor, had no inhibitory activity against CYP1A2, 2A6, 2E1 and 2J2, while it showed an inhibitory effect against CYP2B6, CYP2C19 and 2D6 with IC50 values of 2.5, 3.6 and 0.5 μ m, respectively. Our findings indicate that acetylshikonin may be a novel general P450 inhibitor, which could replace SKF-525A [1].

Reversible inhibition study [1]
All incubations were performed in triplicate, and the data are presented as average values. The inhibitory effect of Acetylshikonin, shikonin and SKF-525A against CYP2J2-mediated astemizole O-demethylase activity was evaluated using pooled HLMs. In brief, the incubation reaction mixtures contained 0.25 mg/ml HLMs, astemizole (1 μ m) and inhibitor (0.5–50 μ m) in 0.1 m m phosphate buffer (pH 7.4) and were pre-incubated for 5 min at 37 °C. The reaction was initiated by the addition of a NADPH-generating system (containing 1.3 m m NADP+, 3.3 m m G6P, 3.3 m m MgCl2 and 500 unit/ml G6PDH). The final volume of the incubation mixture was 100 μl. After a 15 min incubation period, the reactions were stopped by adding ice-cold acetonitrile containing 15 ng/ml terfenadine as the internal standard (IS). After centrifugation, aliquots (1 μl) were injected into a liquid chromatography–tandem mass spectrometry system (LC–MS/MS) as described previously (Lee, Wu, & Liu, 2014).
To evaluate the inhibitory activity of Acetylshikonin, shikonin and SKF-525A against nine other P450 isoforms, namely CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A, a previously developed substrate cocktail method was used (Joo & Liu, 2013; Kim et al., 2005). The substrate concentration, the selected reaction monitoring (SRM) transitions, and the collision energies determined for each metabolite are listed in Table 1. Following a 15 min incubation of HLMs (0.25 mg/ml) in the presence or absence of the inhibitor, the reaction was terminated and the mixtures were centrifuged. Aliquots of the supernatants were analysed by LC–MS/MS as described previously (Joo & Liu, 2013; Kim et al., 2005), with some modifications. The inhibitory effect of acetylshikonin was also evaluated for CYP3A-mediated testosterone and nifedipine metabolism as described previously (Lee, Shon, & Liu, 2016).

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Time-dependent inhibition study [1]
The time-dependent inhibition (TDI) was measured using an IC50 shift method. The HLMs (0.25 mg/ml) were pre-incubated with the inhibitor (Acetylshikonin or thelephoric acid) at five different concentrations (0.5–50 μ m) in the presence of an NADPH-generating system for 30 min. Thelephoric acid, a known non-specific and time-dependent P450 inhibitor (Song et al., 2014), was used as a positive control. The reaction was initiated by adding P450 substrate, the samples were further incubated for 15 min, and then the reaction was terminated by the addition of 100 l ice-cold acetonitrile containing IS. After centrifugation, aliquots of the supernatants were analysed by LC–MS/MS.

[1]. Acetylshikonin is a novel non-selective cytochrome P450 inhibitor. Biopharm Drug Dispos. 2017 Dec;38(9):553-556.

Acetylshikonin is an acetate and hydroxy-1,4-naphthoquinone. It has been reported to exist in plants of the genera Arnebia decumbens and Arnebia euchroma, as well as other organisms with relevant data. However, acetylshikonin exhibited strong inhibitory activity against all tested P450 enzymes, with IC50 values less than 5.0 μM. These results indicate that the compound exhibits similar inhibitory activity against all ten P450 enzymes, with IC50 values ranging from 1.4 to 4.0 μM (Table 2), and its inhibitory effect is stronger than known nonspecific P450 inhibitors such as shikonin (2.0 μM ≤ IC50 ≤ 11.4 μM) and the commonly used SKF-525A. Acetylshikonin also inhibits CYP3A-mediated metabolism of testosterone and nifedipine, with IC50 values of 5.2 μM and 3.0 μM, respectively, indicating that it inhibits CYP3A activity in a substrate-independent manner. These experiments demonstrated that, in human liver microsomes (HLM), acetylshikonin exhibited stronger non-specific inhibitory activity against these ten P450 isoenzymes than SKF-525A and shikonic acid. Similar to acetylshikonin, its structural analog shikonin also showed strong inhibitory activity against the ten P450 isoenzymes (IC50 < 5.2 μM, Table 2). Next, this study investigated whether the presence of NADPH would lead to changes in IC50 values (Table 2). In the presence of the NADPH-generating system, the inhibitory activity of acetylshikonin against the ten P450 isoenzymes (0.7 μM ≤ IC50 ≤ 1.8 μM) was similar to that in untreated human liver microsomes (HLM) (0.9 μM ≤ IC50 ≤ 3.3 μM), indicating that acetylshikonin is not a time-dependent inhibitor. Acetylshikonin (5 μM) showed inhibition rates exceeding 60% against all tested P450 isoenzymes (Figure 1), while SKF-525A showed inhibition rates below 10% against any of its target isoenzymes (CYP1A2, CYP2A6, CYP2C9, and CYP2E1). In previous studies, acetylshikonin inhibited CYP2J2-mediated astemizole O-demethylase activity non-competitively (Park et al., 2017). Therefore, it is hypothesized that acetylshikonin may also inhibit the other nine P450 enzymes non-competitively. In summary, this study evaluated the inhibitory potential of acetylshikonin against P450 isoenzymes. The results indicate that acetylshikonin strongly inhibits the activity of ten P450 isoenzymes in an NADPH-independent manner. These results suggest that acetylshikonin could be used as a novel nonspecific P450 inhibitor, replacing SKF-525A or shikonin, in studies using human liver microsomes (HLM) for response phenotypic analysis. [1]

C18H18O6

330.331925868988

330.11

54984-93-9

Acetylshikonin;24502-78-1

32464

Typically exists as solid at room temperature

2.691

2

6

5

24

599

0

O(C(C)=O)C(C/C=C(\C)/C)C1=CC(C2C(=CC=C(C=2C1=O)O)O)=O

WNFXUXZJJKTDOZ-UHFFFAOYSA-N

InChI=1S/C18H18O6/c1-9(2)4-7-15(24-10(3)19)11-8-14(22)16-12(20)5-6-13(21)17(16)18(11)23/h4-6,8,15,20-21H,7H2,1-3H3

[1-(5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl] acetate

Acetylshikonin; 54984-93-9; DL-Acetylshikonin; 24502-78-1; [1-(5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl] acetate; Shikonin, acetyl; CHEBI:81069; 1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl acetate;

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.)