1. Introduction
Colorectal cancer (CRC) is a malignant tumor of the gastrointestinal tract that is highly likely to metastasize and recur, and it is associated with an extremely high mortality rate [
1]. Colorectal adenoma (CRA) is a frequent intestinal mucosal disease, which can progress into CRC in the adenoma-carcinoma sequence [
2]. Early chemopreventive interventions of precancerous CRA are the most effective measure for the reduction in mortality and morbidity associated with CRC [
3]. Currently, nonsteroidal anti-inflammatory drugs, COX-2 inhibitors, and calcium are potential candidates as chemopreventive agents for CRC. However, the limitation of a lower effectiveness and side effects hindered further application [
4].
3,4,5,4’-Trans-tetramethoxystilbene (Synonyms: DMU-212,
Figure 1) is a methylated analogue of resveratrol possessing greater bioavailability and stronger anti-cancer activity [
5]. It has been examined as a potential chemopreventive agent that can prevent CRC progression through reducing the number of adenomas and suppressing tumor formation in the colon of the Apc
Min/+ mice; the inhibitory effect of DMU-212 on CRA growth may be related to the interference with prostaglandin E-2 generation in cells [
6]. In a previous study of ours, we demonstrated that the mechanisms of CRA prevention using DMU-212 were related to gut microbiota and its metabolites (unpublished data) and, thus, DMU-212 has the potential for use as a chemopreventive agent to prevent CRC. Despite the outstanding efficacy of DMU-212 for anti-CRA, the active constituents and in vivo metabolism study of DMU-212 related to the pharmacological effect are uncertain.
According to one study, DMU-212 undergoes metabolic oxidation, hydroxylation and O-demethylation reactions in liver extracts of mice and an incubate of mouse liver microsomes, as a result of which five metabolites are generated: DMU-214, DMU-281, DMU-291, DMU-295 and DMU-807. DMU-212, administrated to mice, produced higher levels of drug in the small intestine, colon mucosae, and brain than resveratrol. Furthermore, DMU-212 is more easily able to cross the blood-brain barrier than resveratrol due to its higher lipophilicity [
7]. It is of utmost importance to identify the metabolites and metabolic pathways of a drug during drug discovery and development. However, taking into highly complex biological matrices, discovering possible metabolites, and identifying the significant features remains a big challenge [
8]. Only a few corresponding metabolites of DMU-212 have been identified using the Quattro Bio-Q tandem quadrupole mass spectrometer upgraded to the Quattro MK II specifications in healthy mice [
7]. This was not sufficient for the research on the active constituents and action mechanisms of DMU-212 for anti-CRA. Consequently, it is necessary to develop efficient strategies that could identify and targeted detect the numerous trace-absorbed prototypes and metabolites from the complex biological matrix to systematically identify and characterize the absorbed components of DMU-212 in CRA spontaneous model Apc
Min/+ mice.
The Orbitrap ID-X Tribrid mass spectrometer with a quadrupole, orbitrap analyzer, and linear ion trap has an enhanced mass resolution power, scan speed, and the ability to perform MSn for better structural characterization. The AcquireXTM data acquisition workflow includes an automatic background subtraction, fast polarity-switching mode, isotope in-tensity-filtering, and other real-time decision-making features. Compound Discoverer 3.3 software carried out the metabolite analysis and compound annotation. Thus, the chemical constituents in complex matrix samples can be detected at trace levels in a sensitive and high-resolution platform. In the present study, a comprehensive strategy was proposed for the rapid identification of the metabolites of DMU-212 after the oral dose of 240 mg/kg for 3 weeks in CRA spontaneous model ApcMin/+ mice based on ultra-high performance liquid chromatography-quadrupole/orbitrap/linear ion trap mass spectrometry (UHPLC-Q/Orbitrap/LTQ MS) combined with the data processing software “Compound Discoverer 3.3” and the AcquireXTM data acquisition workflow. The representative active metabolites were supported by data from molecular docking assays. This study provides valuable information regarding DMU-212’s active constituents and its mechanism of action for preventing CRA.
3. Discussion
Identifying drug metabolites can be crucial to finding potential drug targets, developing safe drug treatments in clinics, and even rationally modifying drugs [
11]. On one hand, for the present drug metabolite identification studies, the components in biological samples and their metabolites are obtained with healthy or disease-model animal-based approaches [
12,
13]. We believe that a core approach to obtaining the active ingredients that serve a therapeutic role can be based on the effectiveness of the medicine [
14]. Only in this case, where a specific medication proves to be efficacious, are the components in biological samples analyzed to clarify the material basis and mechanism of action for the drug’s efficacy. On the other hand, in order to identify metabolites and to better understand the structure of drugs, high-quality tandem mass spectra are essential [
15]. Without previous experience and prediction of metabolic pathways, identifying low abundant metabolite ions in in vivo samples with a complicated biological matrix presents a substantial challenge [
8]. In this study, UHPLC-Q/Orbitrap/LTQ MS combining AcquireX
TM, a novel data-dependent acquisition workflow, was used to analyze DMU-212 metabolic profiles in vivo based on the effectiveness of the medicine. Through the AcquireX
TM data acquisition workflow, background subtraction and method updating were performed in real-time. Hence, only the MS
2 of potential metabolites were triggered using the updated method without any user intervention. In total, 63 DMU-212 metabolites were identified after the oral dose of 240 mg/kg for 3 weeks in CRA spontaneous model Apc
Min/+ mice, including 48 metabolites in the intestinal contents, 48 metabolites in the liver, 34 metabolites in the serum, and 28 metabolites in the colorectal tissues, centering on the further reaction of the prototype, demethylated DMU-212 and oxidized DMU-212.
As we know, before oral drugs can be used to enter the blood circulation in the body, they must first be digested in the gastrointestinal tract in order to be absorbed in a suitable form [
16]. As a result, the potential metabolic role of gut microbiota on drug metabolism is gaining increasing attention [
17,
18,
19,
20]. Our research found that the gut microbiota may be an important site of DMU-212 metabolism in vivo, and DMU-212 mainly undergoes rapid Phase I, such as demethylation, desaturation, dehydration and oxidation, and Phase II, including methylation metabolism, in the gut microbiota. There is increasing evidence that the gut microbiota affects drug metabolism by altering the structural and functional properties of drugs, which is primarily mediated by unique enzymes encoded within the microbiome [
21,
22,
23]. That DMU-212 is being metabolized by gut microbiota may also have something to do with the fact that the gut microbiota contain a wide range of enzymes. Firmicutes and Bacteroidetes were the dominating taxa in the gut microbiota, which have many enzymes, such as oxidase, reductase, and esterase, allowing for many catalytic processes such as oxidation (hydration, hydrogenation, hydroxylation, methylation, oxygenation), and reduction (dehydration, dehydroxylation, demethylation) [
24,
25]. After being processed by gut microbes, most drugs, including DMU-212, are reabsorbed by the intestinal epithelium and then enter the enterohepatic circulation. In addition to being the main metabolic organ, the liver is also the main site of drug metabolism. As we expected, 48 metabolites were observed in the Apc
Min/+ mice’s liver, which inferred that the liver possessed high metabolic activity for DMU-212. In the liver, CYP450 plays an important role in drug metabolism [
26]. A previous study demonstrated that via CYP1A1, DMU-212 may be demethylated to DMU-218 and oxidized to DMU-214, and the biological activity of the parent compound may be dependent on its conversion to DMU-214 and the level of this enzyme [
27]. In contrast, our results showed that DMU-212 not only included demethylation and oxidation in the Apc
Min/+ mice’s liver but was also involved in many other reactions including Phase I, such as desaturation and dehydration, and even Phase II reactions, such as acetylation, glucuronide and cysteine conjugation, which were responsible for the identification of abundant DMU-212 metabolites. In general, the drug is metabolized in the liver and then distributed into the blood. During previous research, DMU-212 was rapidly cleared from the blood within an hour after administration, and a small amount of the prototype was detected [
7]. In this paper, in the serum of the Apc
Min/+ mice orally administered with DMU-212, the parent drug was also low, which indicated that DMU-212 was metabolized by intestinal bacteria or was absorbed into the blood and underwent hepatic first-pass elimination, followed by Phase I and II metabolism to be quickly transformed into the other metabolites, especially the demethylated, dehydrated and oxidized metabolites involved in Phase I, and the acetylated, glycine/glucuronide-bound metabolites involved in Phase II observed in this paper.
Medicines enter the bloodstream and are delivered to the target tissue where they exert their therapeutic impact. Colorectal tissue is the target tissue in which DMU-212 might prevent malignancy or delay its onset. In our study, in addition to the most abundant parent drug detected in the Apc
Min/+ mice’s colorectal tissue, demethylated, desaturated and oxidized metabolites were also found. In addition, the oxidized and cysteine-bound metabolite M54 was also a major Phase II metabolite and found at a relatively high level in colorectal tissue. Phase II reactions convert compounds to more water-soluble and often less active or toxic derivatives to increase excretion [
28]; therefore, metabolite M54 may not be the pharmacologically active constituent. Bile acid metabolism associated with gut microbiota may contribute to the pathogenesis of colon cancer. In addition, our recent findings underline the regulatory roles of DMU-212 in dysregulated gut microbiota and BA metabolism to prevent CRA in Apc
Min/+ mice (our unpublished data). Microbial BSHs, which initiate bile acid metabolism, are highly related to CRC, these enzymes have been considered a promising target in the manipulation of gut microbiota to benefit human health [
9]. Therefore, we further explored the possible molecular interactions of the metabolites obtained in the colorectal tissue with BSHs using molecular docking experiments. Molecular docking showed that the target BSHs had a strong binding activity with the main metabolites. In addition to the reported five in vivo metabolites [
7], several novel demethylated and/or oxidized metabolites have been characterized from the Apc
Min/+ mice’s colorectal tissue, which may affect bile acid metabolism to prevent CRA by acting on the BSHs’ target. These provided a hint that DMU-212 metabolic products may be the pharmacodynamic material basis of DMU-212 to prevent CRA. However, it is still necessary to conduct further experiments to confirm this speculation.
4. Materials and Methods
4.1. Chemicals and Reagents
The resveratrol analogue DMU-212 was synthesized according to the reference method [
29]. Its purity (>98%) (
Figures S1 and S2) and structure were determined using nuclear magnetic resonance spectroscopy (
Figure S3,
Table S1). Methanol, formic acid and acetonitrile (HPLC grade) were purchased from Fisher Scientific (Fisher Scientific, Waltham, MA, USA).
4.2. Animal Experiments
All of the experiments were approved by the Animal Care Welfare Committee of the Heilongjiang University of Chinese Medicine. In total, 20 ApcMin/+ mice (males aged 4 w) were obtained from GemPharmatech Co. Ltd. (Nanjing, China) and housed under a standard 12-h light-dark cycle at 25 ± 2 °C and 60 ± 5% humidity with free access to water and a high-fat diet (Research Diets, D12492; 60% fat by calories). At 7 weeks of age, the ApcMin/+ mice were randomly assigned to the vehicle-treated ApcMin/+ (MOD) and DMU-212-treated ApcMin/+ groups (DMU) (10 mice per group). The MOD group was orally administrated 0.5 % sodium carboxymethyl cellulose (CMC-Na) per day. The DMU group was orally administrated 240 mg/kg of DMU-212 per day. Drug treatment was performed once daily for 28 successive days. The efficacy of DMU-212 prevention for CRA was evaluated using blood feces, colon length, spleen index, adenoma number, histopathology and inflammatory cytokines, and CRA-related protein expression in colonic tissues. The data is being published in detail in a different publication.
4.3. Collection and Preparation of Biological Samples
Blood, liver, colorectal tissues and intestinal contents samples were collected at 1 h after the last administration. The serum was obtained through the centrifugation of blood in a refrigerated centrifuge at 3000 rpm (4 °C). A 300 μL volume of the serum sample was added to 900 µL of methanol and vortexed for 1.0 min to precipitate the protein and the sample was centrifuged at 12,000 rpm and 4 °C for 10 min. The supernatants were then concentrated using a speed vacuum concentrator and the residue was redissolved using 100 µL of methanol and centrifuged at 12,000 rpm for 10 min. In total, 0.1 g intestinal contents were collected and resuspended in 1 mL of methanol followed by centrifugation (12,000× g rpm, 4 °C, 10 min). The supernatants were dried in vacuo and resuspended in 200 μL of methanol and centrifuged at 12,000 rpm for 10 min. A total of 0.2 g of the mice’s liver tissue and colorectal tissues were weighed, respectively, and homogenized in 1 mL of methanol, and then centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was concentrated in vacuo and redissolved in 200 µL of methanol, and centrifuged at 4 °C at 12,000× g rpm for 10 min. Finally, the supernatants collected from the four kinds of samples were passed through a 0.22 μm membrane filter and a 2 μL sample was injected into the UHPLC-Q/Orbitrap/LTQ MS system for analysis.
4.4. UHPLC-Q/Orbitrap/LTQ MS Analysis
The liquid chromatographic separations were performed on a Vanquish (Thermo Fisher Scientific, Waltham, MA, USA) system hyphenated with a Q/Orbitrap/LTQ mass spectrometer system (Thermo Fisher Scientific). As a stationary phase, a Waters Corporation ACQUITY UPLCTM BEH C18 column (100 mm × 2.1 mm, i.d., 1.7 microns) was used. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The gradient was established as follows: 0–2 min, 1–10% B; 2–4 min, 10–40% B; 4–5 min, 40–65% B; 5–12 min, 65–100% B; 12–13 min, 100% B; 13–14 min, 100–1% B; and 14–17 min, 1% B. The flow rate was 0.3 mL/min and the column temperature was set at 35 °C.
A heated electrospray ionization interface, working in positive mode, was used in the mass spectrometer. The parameters were the following: spray voltage of 3.5 kV; sheath gas of 40 arb; and auxiliary gas of 15 arb. The ion transfer tube and the vaporizer temperature were at 350 °C. The scanning mode was full MS/DD-MS2 based on the AcquireXTM intelligent data acquisition technology (Thermo Fisher Scientific), with an Orbitrap resolution of 120,000 and a mass range of m/z 50–1200.
4.5. Data Processing Software
The Xcalibur 4.0 workstation software (Thermo Fisher Scientific) was used for raw data recording and processing. Using Compound Discoverer 3.3 (Thermo Fisher Scientific), post-processing of the data was performed to extract the metabolite-related datasets based on the structural correlation between the drug and its metabolites. A maximum tolerance of 5 ppm was set for the mass error. Using blank samples, the workflow subtracted the chemical backgrounds, aligned the retention times, and found the expected compounds and metabolites. Fragment ion search (FISH) scoring was used, and each annotation was then manually evaluated based on the HCD, DDA spectra, molecular formula, and FISH coverage.
4.6. Molecular Docking
The 3D structure of bile salt hydrolases (BSHs) was downloaded from the RCSB PDB (
https://www.rcsb.org/, accessed on 18 April 2023). The PubChem database (
https://pubchem.ncbi.nlm.nih.gov/, accessed on 18 April 2023) was used to download the SDF format files of the 2D structure of DMU-212. The structures of another core active component were made with ChemDraw (
http://www.perkinelmer.com/category/chemdraw, accessed on 18 April 2023). Molecular docking with the main active components of the main active ingredients and BSHs was performed using PyMoL 2.3.0 and AutoDock Vina 18. The binding activity was assessed using binding energy.
5. Conclusions
In this paper, the metabolic profiles of DMU-212 in ApcMin/+ mice’s serum, liver, colorectal tissues and intestinal contents were systematically and comprehensively investigated based on the effectiveness of the medicine. A total of 63 DMU-212 metabolites were determined and summarized through quick, sensitive and accurate UHPLC-Q/Orbitrap/LTQ MS combined with the data processing software “Compound Discoverer 3.3” and the AcquireXTM data acquisition workflow. In addition, further verification of the representative active metabolites was employed using molecular docking analysis. The Phase I metabolites of DMU-212 were mainly produced via demethylation, oxidation, desaturation, dehydration, reduction and hydration, while the major Phase II metabolites were methylation, acetylation, glucuronide, cysteine, glycine and glutamine conjugation products. As a result of this study, a simple method for studying drugs’ metabolism in vivo is provided, as is scientific and reliable support for a complete understanding of DMU-212’s metabolism and transformation. In addition, as a reference for the further development of new drugs, this is also important for a deeper understanding of the active constituents of DMU-212 and its action mechanisms for CRA prevention.