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 ester and a hydroxy-1,4-naphthoquinone.
Acetylshikonin has been reported in Arnebia decumbens, Arnebia euchroma, and other organisms with data available.

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However, acetylshikonin exhibited strong inhibitory activities against all P450 enzymes tested, with IC50 values < 5.0 μ m. These findings showed that the compound had similar inhibitory potential against all ten P450 enzymes in the range 1.4–4.0 μ m (Table 2), and its inhibitory effects were stronger than those of thelephoric acid, a well-documented non-specific P450 inhibitor (2.0 μ m ≤ IC50 ≤ 11.4 μ m), and the commonly used SKF-525A. Acetylshikonin also inhibited CYP3A-mediated testosterone and nifedipine metabolism with IC50 values of 5.2 and 3.0 μ m, respectively, indicating that it inhibits CYP3A activity in a substrate-independent manner. These experiments demonstrated that acetylshikonin is a more potent non-specific inhibitor of these ten P450 isoforms than are SKF-525A and thelephoric acid in HLMs. Similar to acetylshikonin, shikonin, a structural analogue of acetylshikonin, showed strong inhibitory effects against ten P450 isoforms (IC50 < 5.2 μ m, Table 2).
The study next investigated whether the presence of NADPH led to shifts in the IC50 (Table 2). The inhibitory potential of acetylshikonin against the ten P450 isoforms in the presence of an NADPH-generating system (0.7 μ m ≤ IC50 ≤ 1.8 μ m) was similar to that in untreated HLMs (0.9 μ m ≤ IC50 ≤ 3.3 μ m), suggesting that acetylshikonin is not a time-dependent inhibitor. Acetylshikonin (at 5 μ m) inhibited all P450 isoforms tested by more than 60% (Figure 1), whereas SKF-525A did not inhibit any of its target isoforms (CYP1A2, CYP2A6, CYP2C9 and CYP2E1) by more than 10%. In a previous study, acetylshikonin inhibited CYP2J2-mediated astemizole O-demethylase activity in a noncompetitive manner (Park et al., 2017). Therefore, acetylshikonin is speculated to inhibit noncompetitively nine other P450 enzymes.
In conclusion, the inhibitory potential of acetylshikonin against P450 isoforms was evaluated. Acetylshikonin strongly inhibited the activities of ten P450 isoforms in a NADPH-independent manner. The results suggest that acetylshikonin can be used instead of SKF-525A or thelephoric acid as a novel non-specific P450 inhibitor in reaction phenotyping studies using HLMs.[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.)