5.1 Materials and reagents

Hydap (purity > 95%) is a 4-hydroxy-2-pyridone alkaloid as one of the prominent products extracted from the sponge-derived Arthrinium arundinis ZSDS1-F3. It was previously separated, purified, and identified by Junfeng Wang’s group [7, 8]. The fermented broth of Arthrinium arundinis was vacuum-condensed and then extracted with EtOAc to yield an EtOAc solution. The EtOAc extract was subjected to silica gel column chromatography and Sephadex LH-20 to obtain the initial purified subfractions. Subsequently, N-hydap was further purified via semipreparative HPLC using an ODS column eluting with 75% MeOH/H2O. Finally, the chemical structure of N-hydap was determined by NMR and HRMS spectra.

The recombinant human CYP isoforms (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP4A11 and CYP4F2, 0.5 nmol) and the recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, 5 mg/mL) were purchased from Cypex (Scotland, UK). The pooled HLMs, pooled HKMs, and pooled HIMs were bought from Corning Incorporated (Corning, NY, USA). The NADPH Regeneration System (including solution A and solution B) was bought from Promega (Madison, WI, USA). Uridine 5′-diphosphoglucuronic acid (UDPGA) was obtained from Sigma-Aldrich (St. Louis, MO, USA). d-Glucuric acid-1,4-monolactone-water, alamethicin, potassium phosphate dibasic (K2HPO4), potassium dihydrogen phosphate (KH2PO4), sucrose, magnesium chloride(MgCl2), EDTA dipotassium salt (EDTA·K2·2H2O), and testosterone (Tes, used as an internal standard, IS) were purchased from Aladdin (Shanghai, China). (2S,3S)-1,4-bis-sulfanylbutane-2,3-diol (DTT), Benzyl methylsulfonyl chloride (PMSF), and (±)-Verapamil hydrochloride (VER, used as an internal standard) were obtained from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Chlorzoxazone (CLP, used as an internal standard) was purchased from MeilunBio® (Liaoning, China). The study used primary antibodies against CYP1A2, CYP2B6, CYP3A4, and UGT1A1 produced by Abcam (Cambridge, MA, USA). Methanol and formic acid were HPLC grade. All analyses were performed in ultrapure water. Regarding the remaining chemicals and solvents, they were of analytical grade or higher quality and utilized as supplied.

5.2 Animal

Animals were used with approval from Southern Medical University’s Ethics Committee in all cases. Male C57BL/6 mice weighing between 22 and 25 g as well as SD rats weighing between 200 and 250 g were obtained from Guangzhou Jinwei Biotechnology Co., Ltd (Guangdong, China). In a pathogen-free environment, the animals were provided with rodent pellets and sterile water, with a 12-h light–dark cycle.

5.3 Preparation of RLMs, RKMs, MLMs, MKMs

In this study, the rats were executed by drawing blood from the abdominal aorta and mice were sacrificed with cervical dislocation before the liver and kidneys were excised. The liver and kidneys were removed, and washed with chilled cleansing solution (contains 8 mM KH2PO4, 5.6 mM EDTA·K2·2H2O, 1 mM DTT and 229.6 μM PMSF), homogenized in a tissue homogenate buffer with a pH of 7.4, (consisting of 1.8 μM KH2PO4, 8 mM K2HPO4, 250 mM sucrose, 1 mM EDTA·K2·2H2O and 229.6 μM PMSF). Subsequently, the homogenate was centrifuged at 10,400 rpm at 4 °C for 15 min, and the supernatant was then centrifuged at 35,000 rpm at 4 °C for 60 min. A 250 mM sucrose buffer was added to the pellet to reconstitute and the resuspension was stored at − 80 °C until use. Microsome protein concentrations were measured by Omni-Easy™ Instant BCA Protein Assay Kit.

5.4 LC–MS/MS analysis

4000 Q TRAP mass spectrometer (AB SCIEX LLC, Redwood City, CA, USA) with an AB SCIEX electrospray ionization (ESI) source was connected to a Nexera X2 LC system consisting of a degasser, binary pump, autosampler, and thermostatic column compartment and operated with AB SCIEX MultiQuant version 3.0.2 software (AB SCIEX LLC, Redwood City, CA, USA). We performed a chromatography separation on a Phenomenex Kinetex XB-C18 column (100 × 2.10 mm, 2.6 μM) using solvents A (0.01% formic acid and 2 mM ammonium formate in water) and B (methanol) as the mobile phase, with gradient elution at 0.4 mL/min. In order to achieve optimal elution conditions, the following conditions must be met: 0–0.3 min, 10% B; 0.3–1.0 min, 10–90% B; 1.0–3.0 min, 90% B; 3.0–3.1 min, 90–10% B; 3.1–6.0 min, 10% B. Column oven temperature was kept at room temperature, and the injection volume was 5 μL. The mass spectrometer was in ESI modes that switch positive/negative ions, and ions were detected by multiple reaction monitoring (MRM) mode. An overview of the MRM transitions and the compound dependent parameters is shown in Supplementary Table 1. The operating conditions of the MS were optimized as follows: Curtain Gas (CUR), 30 psi; Collision Gas (CAD), medium; Ion Spray Voltage (IS), 5500 V; Temperature (TEM), 550 °C; Ion Source Gas1 (GS1), 55 psi; Ion Source Gas2 (GS2), 55 psi.

5.5 Method validation

To verify the feasibility and reliability of the established method, studies on the linearity, accuracy, precision, and stability of N-hydap in potassium phosphate buffer (KPI, pH 7.4, were prepared by mixing KH2PO4 and K2HPO4 in corresponding proportions, and then adding NaOH and H3PO4 to pH 7.4), plasma, and tissue homogenate were carried out. The peak area ratios (analyte/IS) were plotted against the concentrations of the analytes prepared to obtain the calibration curves. Least squares linear regression analysis was used to determine the slopes, intercepts, and determination coefficients (R2) which should be 0.99 or above of calibration curves. The standard curve equations were determined as y = 0.0001512x + 0.04394 (R2 = 0.9980) for KPI, y = 0.001579x − 0.09233 (R2 = 0.9923) for plasma, and y = 0.002271x + 0.03542 (R2 = 0.9978) for tissue homogenate (Supplementary Fig. 1A–C).

To determine the accuracy and precision of the analytes within and between days, five samples were spiked at three quality control concentrations, either on the same day or on 3 consecutive days. Accuracy (%) was calculated by determining the percentage deviation from the theoretical concentration [(observed value of concentration)/(true value of concentration) × 100%]. Precision (%) was obtained by calculating the relative standard deviation (% RSD) for inter- and intra-day replicates. These precisions remained within the acceptable 15% limit and all measurements demonstrated satisfactory accuracy, with recovery percentages ranging from 85 to 115% of the reference value, as shown in Supplementary Table 2.

The stability of the KPI solution was examined after storage at 4 °C for 24 h and incubation at 37 °C for 4 h, which was conducted using the QC samples at three concentrations (five samples for each concentration). Stability in plasma and tissue homogenate was assessed by testing samples stored at both 4 °C and room temperature for 24 h. The results of the stability experiments indicated no significant degradation (Supplementary Table 3). In conclusion, the standard curves, inter- and intra-day precision, accuracy, and stability all met the required criteria for working with KPI solution, plasma, and tissue homogenate.

5.6 Incubation of N-hydap with microsomes

Various microsomes including HLMs, HKMs, HIMs, RLMs, RKMs, MLMs and MKMs were involved in CYP and UGT reactions of N-hydap. A typical CYP incubation mixture included 0.5 mg/mL microsomes protein, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, 0.4 U/mL glucose-6-phosphate dehydrogenase, and N-hydap (1, 2.5, or 10 μM) in the 50 mM potassium phosphate buffer (KPI, pH 7.4) [35]. The incubation mixture was preincubated at 37 °C for 5 min, and the reaction was started by adding NADP to form a NADPH-regenerating system. Simultaneously, the classic UGT reaction system consisted of 50 mM KPI, 5 mM MgCl2, 0.5 mg/mL microsomes, 0.125 mg/mL alamethicin, and 5.25 mg/mL saccharolactone. The reaction was initiated after supplying UDPGA. The CYP and UGT mixtures were incubated at 37 °C in a shaking water bath (80 rpm) for 1.5 h to enhance metabolite formation. To terminate the reaction, an ice-cold solution of Tes (50 ng/mL, dissolved in methanol and used as an internal standard) was added to half of the mixture volume. The mixture was vortexed for 30 s and then centrifuged at 13,000 rpm for 15 min to eliminate any precipitated protein. The resulting supernatant was transferred to autosampler vials, and 5 μL was injected into the LC–MS system. All reactions were conducted in triplicate.

5.7 Assay for metabolic stability in HLMs

The identical concentration of HLMs was utilized for both the CYP reaction and UGT reaction. At predetermined time intervals of 0, 15, 30, and 60 min, 100 µL samples were taken from the reaction volumes. To halt the reaction, 50 µL of 50 ng/mL Tes was added. Samples were analyzed using LC–MS to compare the remaining level of N-hydap between the CYP reaction and UGT reaction. T1/2 and CLint (in vivo) (mL/min/kg) were calculated as reported previously [36]:

where k represents the slope of the line derived from plotting the natural logarithm of the percentage (Ln %) of N-hydap remaining in the reaction mixture against the incubation time (minutes).

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5.8 Metabolism of N-hydap in recombinant human CYP and UGT enzymes

Incubation of N-hydap (2.5 μM) with eight recombinant human CYP enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP4A11 and CYP4F2) at three enzyme concentrations (2, 4, and 20 μg/mL) was conducted following the same procedure as described above for microsomes. Subsequently, incubation with N-hydap (1, 2.5, and 6 μM) and twelve recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17) was performed, with the enzyme concentration used at 2 μg/mL. All reactions were carried out in triplicate.

5.9 Kinetics of N-hydap glucuronidation in microsomes and UGTs

The substrate concentrations used for HLMs were 0.05, 0.5, 2, 2.5, 5, 10, 25, 50, and 100 μM. For HKMs, HIMs, RKMs, MLMs, and MKMs, the substrate concentrations used were 0.05, 0.1, 0.2, 0.4, 0.8, 1, 2, 2.5, 10, 25, 50, 100, and 200 μM. For RLMs, the substrate concentrations used were 0.05, 0.1, 0.2, 0.4, 0.8, 1, 2, and 2.5 μM. Lastly, for UGT1A1 and UGT1A3, the substrate concentrations used were 0.05, 0.25, 0.5, 1, 2, 2.5, 5, 10, 25, 50, 100, and 200 μM. Incubation time for all concentrations was 1 h. The rates of N-hydap-G by enzymes were expressed as nmol/mg/min, representing the amount of metabolites formed per minute per milligram of protein. Kinetic parameters were determined by analyzing the Eadie–Hofstee plots. Each incubation was performed in triplicate and repeated independently three times.

5.10 Pharmacokinetics studies

Mice were fasted for 12 h with access to water before drug administration. N-Hydap was dissolved in a solution containing 50% methanol and water. Before administration, the drug was diluted with PBS to achieve concentrations of 5 mg/kg and 2 mg/kg for gastric gavage and tail vein administration, respectively. Blood samples (120 μL) were collected from the orbital venous plexus at 2, 5, 10, 15, 30, 60, 90, 120, 180, and 240 min after N-hydap administration. The blood samples were collected into heparinized micro-centrifuge tubes and immediately centrifuged at 8000 rpm for 5 min at 4 °C. Plasma samples were prepared by adding an internal standard (IS) solution, Tes to the samples, followed by vortexing. The mixtures were then centrifuged at 4 °C and 13,000 rpm for 15 min. The resulting supernatant was dried under vacuum for 3–4 h, and the dry powder was reconstituted with a solution of 50% methanol and water. Finally, the plasma samples were analyzed using LC–MS, following the previously described method.

5.11 Distribution studies

The heart, liver, spleen, lungs and kidneys were removed from mice after administering a dose of 5 mg/kg through oral gavage and 2 mg/kg through tail vein injection at 30 min and 240 min, followed by homogenization with normal saline. The resulting homogenate was centrifuged at 4 °C, 13,000 rpm for 20 min, and the supernatant was then mixed with the internal standard by vortexing. The mixtures were centrifuged at 4 °C, 13,000 rpm for 15 min. The resulting supernatant was then subjected to vacuum drying for 3–4 h. Lastly, the dried powder was reconstituted with a mixture of 50% methanol and water, and then centrifuged for LC–MS testing.

5.12 Drug metabolic enzyme activity in MLMs and MKMs

Mice were randomly divided into four groups, with eight animals in each group. The mice received oral pretreatment with N-hydap for 7 days at doses of 2, 5, and 10 mg/kg/day, which were considered as the low dose, medium dose, and high dose, respectively. Correspondingly, the control group received PBS. On the 8th day, the liver and kidneys were removed, and organs from eight mice per group were combined to prepare MLMs and MKMs using the detailed procedures above. Subsequently, we determined the activity of CYP and UGT enzymes by specific substrate metabolized with MLMs (0.5 mg/mL) and MKMs (0.2 mg/mL) in vitro. Specifically, the following selective substrates were used for the respective enzymes: PH (1 μM) for CYP1A2, DIC (1 μM) for CYP2C9, MP (90 μM) for CYP2C19, DM (1 μM) for CYP2D6, CZX (10 μM) for CYP2E1, Tes (1 μM) for CYP3A4, SN38 (1 μM) for UGT1A1, CACD (100 μM) for UGT1A3, Sero (20 μM) for UGT1A6, and Prop (1 μM) for UGT1A9. All substrates were dissolved in methanol or DMSO. The incubation procedure was carried out as mentioned above, and the reaction was terminated with VER (20 ng/mL) in ESI+ mode and CLP (200 ng/mL) in ESI− mode. In the ex vivo experiments, the activity of enzymes was measured by quantifying the production of various metabolites (nM/mg protein/min). The metabolites and their corresponding enzymes are as follows: APAP from PH, 4-OH-DIC from DIC, 4-OH-MP from MP, DXO from DM, 6-OH-CZX from CZX, 6-OH-Tes from Tes, SN38-G from SN38, CACD-G from CACD, Sero-G from Sero, Prop-G from Prop. The measurements were performed using multiple reaction monitoring (MRM) transitions, which are specific mass spectrometry transitions used for quantification. The optimized parameters for each metabolite, such as declustering potential and collision energy, were described in Supplementary Table 1. All experiments were conducted in triplicate, to ensure accuracy and reproducibility.

5.13 Real-time PCR analysis

Total RNA from liver and kidney samples taken after 7 days of N-hydap gavage was extracted using the Animal Total RNA Isolation Kit (Foregene, Chengdu, Sichuan, China) following the manufacturer’s instructions. The concentrations of total RNA were determined by a nucleic acid concentration tester at 260/280 nm. A HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, Jiangsu, China) was used to measure the concentration of the synthesized cDNA. ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, Jiangsu, China) and detection were performed using a Light Cycler 480 II (ROCHE, Basel, Switzerland). Real-time PCR was used to analyze the relative mRNA levels of Cyp1a2, Cyp2b10, Cyp2c39, Cyp2d22, Cyp2e1, Cyp3a11, Ugt1a1, Ugt1a6a and Ugt1a9 (human homologs: CYP1A2, CYP2B6, CYP2C19, CYP2D6, CYP2E1, CYP3A4, UGT1A1, UGT1A6, and UGT1A9) in the liver, kidneys, and intestines. The sequences of the primers used in the experiment are provided in Supplementary Table 4. The relative mRNA levels of the target gene were normalized against the levels of GAPDH mRNA.

5.14 Western blot analysis

After 7 days of N-hydap gavage, the liver and kidney tissues were lysed using RIPA buffer supplemented with a 1% protease inhibitor cocktail. Protein concentrations were tested using a BCA estimation kit following the instructions provided by the manufacturer. Then the protein samples were mixed with 5× loading buffer and the mixture was denatured at 100 °C for 10 min. Equal amounts of protein (24 μg) were separated by 10% SDS-PAGE and subsequently transferred from the gel to the PVDF membrane. After blocking for 1–2 h with blocking fluid containing 0.1% Tween-20 (TBST), the corresponding primary antibodies were diluted according to the instructions and incubated with the membrane at 4 °C overnight. The membrane was washed before incubation with the corresponding secondary antibody at a dilution of 1:10,000 for 1–2 h at room temperature. Western blot signals were detected using an ECL chemiluminescence detection agent. The relative intensity of each protein band was scanned and quantified using Image J software.

5.15 Data analysis

DAS 2.0 was used to calculate pharmacokinetic parameters using the standard non-compartmental method. All results were presented as mean ± standard deviation and significant differences were analyzed using One-way ANOVA by GraphPad Prism 8. Statistical significance was defined as p < 0.05.