Chemicals and reagents

AT7519 and a corresponding deuterated standard [2H]8-AT16043M (d8-AT7519) were supplied as a gift by Astex Pharmaceuticals (Cambridge, UK), as methane sulfonic acid salts. APAP was purchased from Apollo (Denton, Manchester, UK) and a [2H]8-APAP (d4-APAP) stock solution was purchased as a certified reference material at 100 µg/mL in methanol from Cerilliant® (Merck, Watford, UK). The structure of APAP, AT7519 and the corresponding isotopically labelled internal standards are shown in Fig. 1.

Fig. 1

Structures of AT7519, acetaminophen (APAP) and their corresponding isotopically labelled standards d8-AT7519 and d4-APAP

Water (LC–MS grade), methanol (LC–MS grade), 2-propanol (LC–MS grade) and acetonitrile (HPLC grade) were purchased from VWR. Formic acid (LC–MS grade) was purchased from Fisher Scientific (Loughborough, UK) and bovine serum albumin 5% in 0.85% sodium chloride from Sigma Aldrich (Dorset, UK).

Instrument and chromatographic conditions

Samples, held at 10 °C in the autosampler, were injected (20 µL) into the chromatographic system of a Waters Acquity Classic UPLC unit with an Acquity UPLC BEH C18 (2.1 × 100 mm 1.7 µm; Waters, Wilmslow, UK) column maintained at 45 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.5 mL/min. Gradient elution was achieved with a total run time of 9.0 min. The gradient conditions were as follows: An initial hold of 5% B for 1.5 min, with an increase up to 95% B over 5 min, held at 95% B for 2 min, followed by a 2 min equilibration at 5% B. Optimised mass spectrometry parameters of de-clustering potential, entrance potential, collision energy and collision cell exit potential for all analytes (APAP and AT7519 and internal standards d8-AT7519 and d4-APAP) are shown in Table 1.

Table 1 Optimized mass spectrometry settings on a QTrap 5500 mass spectrometer for APAP, AT7519 and isotopically labelled internal standards, operated in positive mode. Voltage (V). All analytes and standards were infused and analysed in positive ion mode

Mass analysis was performed on a QTrap 5500 triple quadrupole mass spectrometer (AB Sciex, Warrington, UK) operated in positive ion electrospray mode (5.5 kV, 500 °C, ion source gas 1 at 60 psi and gas 2 at 40 psi). In multiple reaction mode, transitions were identified (see results), and the peak area ratio of APAP/d4-APAP and AT7519/d8-AT7519 were used to calculate the amount of APAP and AT7519 by linear regression analysis of a calibration curve (0.5–50 ng/mL). LC–MS/MS data were evaluated by investigators blinded to the sample treatment.

Preparation of drug solutions, calibrations standards and quality control samples

Stock solutions of APAP, AT7519 and d8-AT7519 were prepared in methanol at a concentration of 1 mg/mL. d4-APAP stock solution was purchased as a certified material at 100 µg/mL in methanol from Cerilliant, UK. A working stock solution containing both APAP and AT7519 was prepared at 50 µg/mL, and further diluted to 0.5, 1, 2.5, 5, 10, 25, 45, and 50 ng/mL. A working internal standard solution containing d4-APAP and d8-AT7519 (50 ng/mL) was prepared in methanol. All standards were prepared in 4% BSA. Each sample was enriched with 1 ng working internal standard. Quality control samples were prepared in 4% BSA solution at four concentrations: LLOQ-QC (0.5 ng/mL), Low-QC (0.75 ng/mL), Mid-QC (5 ng/mL) and the High-QC (40 ng/mL). To each QC sample, 1 ng working internal standard was added. All standards, sample and QC-samples were defrosted and mixed for 10 min at 400 rpm before extraction. All standard solutions were stored at –20◦C and brought to room temperature before use.

Sample preparation

Sample and calibration standard extractions were automated on a 96-well protein precipitation (PPT +) plate (Biotage, Sweden). Frozen samples were thawed at room temperature and 50 µL of serum was transferred to a 2 mL 96-well plate. Standards were prepared in 50 µL of 4% BSA. Samples and standards were enriched with the working internal standard (1 ng d4-APAP and d8-AT7519) and diluted in water (1:1). Samples and calibration standards were transferred to an Extrahera liquid handling robot (Biotage, Uppsala, Sweden). The liquid handling robot added acetonitrile (400 μL) to each well then transferred the samples to a PPT + plate (2 mL, Biotage) for extraction. The eluate was pulled through under positive pressure into a 2 mL deep well 96 well collection plate (2 mL, Waters, UK).

The eluate was reduced to dryness under nitrogen stream at 40 °C on an SPE Dry 96 Dual Sample Concentrator (IST, UK). Once dry, samples were resuspended in water:methanol (90:10 v/v; 100 µL) and sealed with a Zone-free 96 well plate sealing film (VWR). The samples were aggregated in a plate shaker (Thermoshaker 3005 GFL, ThermoScientific, UK) for 10 min at 600 rpm prior to analysis by LC–MS/MS.

Assessment of accuracy and precisionAccuracy

To assess the accuracy of the method, QC samples were prepared using 4% BSA solution and spiked with known amounts of AT7519 and APAP. The QC samples were analysed against the calibration curve. Here, the accuracy of both the standards and QC samples are reported as a percentage of the nominal value (%NOM). The %NOM is the calculated concentration expressed as a percentage of the nominal concentration using the following equation.

$$accuracy\left(\%NOM\right)=\frac\;\times\;100$$

To assess the accuracy of the standard curve, the concentrations of each standard were back calculated by plotting the peak area ratios of AT7519 and APAP to the internal standards d8-AT7519 and d4-APAP respectively. A weighted (1/x2) least-squares linear regression analysis was performed using Analyst® 1.7.1 software. Acceptance criteria for accuracy of both standards and QC samples was set at ± 15%, except the LLOQ standard and LLOQ QC samples, which had an acceptance of ± 20%.

Intra batch (within-run) accuracy

To assess within run (intra-batch) accuracy, alongside the standard curve; four QC sample replicates per level (LLOQ, Low, Medium and High); 0.5, 0.75, 5.0 and 40.0 ng/mL) were included. The following equation was used to calculate the within-run (intra-batch) accuracy of each QC level.

$$\mathrm=\frac \times 100$$

Inter batch (between run) accuracy

To assess the between run accuracy (inter-batch), three batches were analysed over two days. Each batch included a standard curve, and four QC sample replicates per level. The inter-batch accuracy of each QC level was calculated using the following equation.

$$\mathrm=\frac \times100$$

PrecisionIntra-batch precision of QC samples

To assess the within run (intra-batch) precision, the coefficient of variation (%CV) of 6 QC levels was calculated in a single batch, using the standard deviation (SD) and arithmetic mean in the following equation, with an acceptance criterion of ± 15%.

$$\mathrm(\mathrm)=\frac \times100$$

Inter-batch precision of QC samples and standards

The between run (inter-batch) precision, was calculated for both the standards (n = 3) and the QC samples (n = 12). The %CV of each standard and QC level was calculated across three batches, using the following equation.

$$\mathrm(\mathrm)=\frac \times 100$$

Specificity and selectivity

Selectivity was assessed by analysing serum samples from mice treated with APAP or AT7519 and vehicle treated mouse samples spiked with either compound separately. Specificity was tested by analysing five serum samples from mice not treated with APAP or AT7519, and five samples from mice that received either AT7519 or APAP.

Recovery and Matrix effects

The recovery of both analytes extracted from BSA using PPT + was assessed. Standards were prepared at 50 ng/mL in 4% BSA (100 μL). Peak areas of standards extracted from BSA (pre-spike, n = 3) were compared to standards spiked into BSA extracts post extraction (post-spike, n = 3). The % recovery was calculated using the following equation.

$$\mathrm=\frac \times100$$

The matrix effect of BSA for both analytes was calculated using analyte peak area in post spike standards (50 ng/mL) (n = 30) in BSA extracts, compared to peak area in a pure solution (n = 3) of the same concentration. The equation to calculate matrix effect is shown below.

$$\mathrm=\frac \times 100$$

Stability

Stability of extracted samples stored at 10 °C in the autosampler was assessed by immediate injections, followed by a second injection of standards and QCs (LLOQ, Low, Mid and High) after 48 h in the autosampler and the peak areas and concentrations compared with analysis at 0 h.

In vivo studies Animal studies

Ten week old C57Bl6J male mice (purchased from Charles River, UK) were acclimatised to unit conditions for 1 week prior to experiments. Mice were housed in groups of five in an individual ventilated cage system, and synchronized to a 10–14 h dark/light cycle with access to food and water ad libitum. All animal experiments were undertaken in accordance with criteria outlined in a license granted under the Animals (Scientific Procedures) Act 1986, and approved by the University of Edinburgh Animal Ethics Committee.

Mice were fasted 12 h prior to a 350 mg/kg injection of APAP in sterile saline, (PanReac Applichem) or vehicle (sterile saline). Standard chow and mash were returned to mice 20 min after injection. To all mice, AT7519 dissolved in sterile saline at 2.5 mg/ml, dosed at 10 mg/kg or vehicle was injected at 16 h post APAP injection and whole blood was collected at 36 h post APAP injection from the caudal vena cava. Whole clotted blood samples were centrifuged at 5,000 g for 5 min, from which the serum supernatant was collected. This process of centrifugation of the collected serum was repeated to ensure no red blood cell contamination. Serum was stored at -80 °C until analysis.

Serum chemistry evaluation

Serum chemistry was performed utilizing a commercial kit (Alpha Laboratories) for alanine aminotransferase (ALT) [27], adapted for use on a Cobas Fara centrifugal analyser (Roche Diagnostics).

Immunohistochemistry

FFPE 4 µm thick sections were dewaxed and rehydrated before heat mediated antigen retrieval for 15 min in either TrisEDTA (Ph9), then permeabilised in PBS 0.1% Tween 20 (PBST). Sections were blocked for 30 min with Protein Block (Spring Bio), then incubated with the primary antibodies HNF4α (1:200, Perseus Proteomics, PP-H1415-00), Ly6G (1:500, Biolegend, 127,602) and minichromosome maintenance complex component 2 (MCM2) (1:200, Cell Signalling, 4007S) overnight at 4 °C. Following washing secondary antibodies; Donkey, anti-goat 555, (Invitrogen a32816), donkey anti-rabbit 647 (Invitrogen A32795) and donkey anti-rat 488 (Invitrogen A21208) were applied for 1 h, with DAPI (1:1000) and mounted with flouromount (Southern Biotech).

Microscopy and Image analysis

Bright field images were also acquired on a Vectra® Polaris™ multi spectral slide scanner (PerkinElmer) and fluorescent images collected on an Operetta CLS High Content Analysis System (PerkinElmer). Numbers and percentage of MCM2 positive hepatocytes (HNF4 α + , DAPI +) cells were analysed using Columbus™ software (Perkin Elmer). On H&E stains, necrotic areas were identified through a lack of intact hepatocyte nuclei, disordered tissue structure, and hepatocyte ballooning. These parameters were used with spectral unmixing to train tissue analysis software, inForm 2.4 (Perkin Elmer) for quantification.

Data analysis

LC–MS/MS data was collected using Analyst® 1.7.1 software and the targeted data and linear regression were evaluated using MultiQuant 3.1.3 (AB Sciex, UK), while assay validation precision and accuracy was calculated in Microsoft Excel ® 2016. Graph Pad Prism 8 was used for statistical analysis of mouse serum APAP and AT7519 concentrations and correlations with tissue damage and repair markers. Testing for normality was completed with a Shapiro–Wilk test. Data without a gaussian distribution and difference in variation were assessed with a Kolmogorov–Smirnov test.