Abstract
Methylation of lysine by histone methyltransferases can be reversed by lysine demethylases (KDMs). Different KDMs have distinct oncogenic functions based on their cellular localization, stimulating cancer cell proliferation, reducing the expression of tumor suppressors, and/or promoting the development of drug resistance. JIB-04 is a small molecule that pan-selectively inhibits KDMs, showing maximal inhibitory activity against KDM5A, and as secondary targets, KDM4D/4B/4A/6B/4C. Recently, it was found that JIB-04 also potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines via the inhibition of a new target, serine hydroxymethyltransferase 2. Pharmacokinetic characterization and an analytical method for the quantification of JIB-04 are necessary for the further development of this small molecule. Herein, a sensitive, specific, fast and reliable UHPLC-MS/MS method for the quantification of JIB-04 in rat plasma samples was developed and fully validated using a SCIEX 6500+ triple QUAD LC-MS system equipped with an ExionLC UHPLC unit. The chromatographic separation was achieved on a reverse phase ACE Excel 2 Super C18 column with a flow rate of 0.5 mL/min under gradient elution. The calibration curves were linear (r2 > 0.999) over concentrations from 0.5 – 1000 ng/mL. The accuracy (RE%) was between −7.4% and 3.7%, and the precision (CV%) was 10.2% or less. The stability data showed that no significant degradation occurred under the experimental conditions. This method was successfully applied to the pharmacokinetic study of JIB-04 in rat plasma after intravenous and oral administration and the oral bioavailability of JIB-04 was found to be 44.4%.
Keywords: JIB-04, LC-MS/MS, validation, Pharmacokinetics
Graphical Abstract
1. Introduction
Histones are highly conserved proteins that play a dynamic role in modulating chromatin structure. Methylation of lysine by histone methyltransferases can be reversed by lysine demethylases (KDMs). A dominating KDM family is formed by JmjC domain containing proteins, containing over 30 members and includes the KDM2 to KDM8 subfamilies [1]. Different KDMs may have distinct oncogenic functions based on their cellular localization. KDM4 proteins are overexpressed or deregulated in multiple cancers, cardiovascular diseases, and mental retardation [2]. KDM4A is a determinant for invasiveness and metastasis in squamous cell carcinoma and ovarian cancer [3–4], KDM4B is highly expressed in ER+ breast cancer [5]. KDM4C is amplified and over-expressed in numerous hematological and solid cancers [6]. KDM5 proteins stimulate cancer cell proliferation, reduce the expression of tumor suppressors, promote the development of drug resistance, the survival of tumor- initiating cells and relapse [7–11]. In human cancer, elevated expression levels of KDM5A were found in hepatocellular carcinoma [12], gastric cancer [13–14] and lung cancer [15–16]. KDM inhibitors are of strong interests as potential therapeutic agents for cancer treatment. Currently available inhibitors suffer from a lack of selectivity and potency.
Both E- and Z-isomers of JIB-04 (Fig. 1) were synthesized along with a series of N-heteroaryl hydrazones derived from aryl N-heteroaryl or bis-N-heteroaryl methanones in search for potential ribonucleotide reductase inhibitors in 1997 [17]. But only when its pan-selective inhibition against KDMs was discovered in 2013 [18–19], the E-isomer of JIB-04 (JIB-04 if without further clarification in the context) started to be noticed. It was also found that the Z-isomer remains inactive. JIB-04 exerts its maximal inhibitory activity against KDM5A, and as secondary targets, KDM4D/4B/4A/6B/4C [18, 20–21]. Since then, JIB-04 as a potential anti-cancer agent has attracted a lot of attention [22]. It is the first epigenetic modulator targeting demethylases to show in vivo action [18]. Recently, it was found that JIB-04 potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines via the inhibition of a new target serine hydroxymethyltransferase 2 (SHMT2) [23]. JIB-04 is known to reduce tumor growth in lung cancer and prostate cancer cell lines [18] and to induce cell death in drug-resistant brain cancer and lung cancer cells [24–25]. It is also known to selectively target colorectal cancer stem cells [26] and Ewing Sarcoma cells [27]. JIB-04 also restores sensitivity to cytarabine in SETD2-mutant leukemia, presumably via inhibition of KDM4 H3K36 demethylase activity [21]. Importantly, JIB-04 has also been shown to have efficacy against taxane/platin-resistant NSCLC [24] and temozolomide- resistant glioblastoma cells [25], and to sensitize tumors to radiation therapy [28].
Fig. 1.
Chemical structures of JIB-04 (E-isomer and Z-isomer) and IS (EPH8).
As the drug development moves forward, pharmacokinetic studies and a sensitive and specific analytical method for the quantification of JIB-04 become inevitably necessary. Until now, only a preliminary biodistribution analysis of JIB-04 in mice has been reported. JIB-04 concentration was determined by a micro-flow LC-time-of-flight (TOF) MS with a LLOQ at 5 nM (equals to 1.545 ng/mL). However, the method has a total of 63-min running and re-equilibration time, no internal standard (IS) was used and no validation was performed [25], thus although a helpful start, the study did not comply with criteria for bioanalytical analysis according to US Food Drug Administration (FDA) and European Medicines Agency (EMA) guidelines [29, 30].
In this study, a sensitive, specific, fast and reliable UHPLC-MS/MS assay for the quantification of JIB-04 in rat plasma samples was developed and fully validated using a SCIEX 6500+ Triple-Quad LC-MS/MS system. The method has a lower limit of quantification (LLOQ) of 0.5 ng/mL in plasma samples with a running time of 3 min. The method has been successfully applied to pharmacokinetic studies of JIB-04 in rats with intravenous (i.v.) and oral administration.
2. Materials and methods
2.1. Chemicals and reagents
JIB-04 (98% E-isomer containing trace Z-isomer) and EPH8 (as internal standard, IS) (Fig. 1) were synthesized according to published methods [17–18]. Dimethyl sulfoxide (DMSO) and Kolliphor® EL were purchased from Sigma-Aldrich (St. Louis, MO, USA) LC-MS grade acetonitrile (ACN), water, and acetic acid were purchased from VWR Chemicals BDH® (Chicago, IL, USA). Blank rat plasma was purchased from Innovative Research (Novi, MI, USA). All other chemicals were of analytical reagent grade and were purchased from commercial suppliers.
2.2. Instrumentation and analytical conditions
The assay was performed on a 6500+ Triple Quad LC-MS/MS System equipped with an ExionLC UHPLC unit (AB SCIEX LLC, CA, USA) and an ACE Excel 2 SuperC18 Column (50 × 2.1 mm, 2 μm, Advanced Chromatography Technologies Ltd., Aberdeen, Scotland, UK). The optimized method used binary gradient mobile phases with water (0.05% acetic acid) as mobile phase A and ACN (0.05% acetic acid) as mobile phase B. A flow rate of 0.5 mL/min was used with 5 μL of injection volume. Running time was 3 min. The time program of the gradient was as follows: Phase B was initially kept at 40% for 0.4 min, increased from 40% to 100% in the following 1.6 min, then kept at 100% for 0.5 min, then decreased from 100% to 40% in 0.3 min and kept stably at 40% for 0.7 min.
Mass spectrometry data were recorded using electrospray ionization (ESI), positive ion detection, and multiple reaction monitoring (MRM) scanning. The ion spray voltage and temperature were set at 5200 V and 550°C, respectively. The curtain gas was set at a flow rate of 50 psi; gas 1(nebulizer gas) and gas 2 (heater gas) were both set at 70 psi. CAD (collision gas) was set at 9. Data was acquired by Analyst software 1.6.3. The MS/MS parameters for JIB-04 and EPH8 are shown in Table 1.
Table 1.
Compound dependent parameters for JIB-04 and the IS
| Q1 (m/Z) (Da) | Q3 (m/Z) (Da) | DP (V) | EP (V) | CE (V) | CXP (V) | |
|---|---|---|---|---|---|---|
| JIB-04 | 309.1 | 230.1 | 70 | 5 | 25.5 | 10 |
| 181.1 | 70 | 5 | 28 | 10 | ||
| EPH8 | 310.1 | 231.0 | 70 | 6 | 25 | 12 |
Q1 = Parent ion, Q3 = Product ion, DP = Declustering Potential, EP = Entrance Potential, CE = Collision Energy, and CXP = Collision Cell Exit Potential.
2.3. Preparation of standard and quality control (QC) samples
A stock solution of JIB-04 (2 mg/mL) was prepared by dissolving 10 mg of JIB-04 in 0.5 mL of DMSO and further diluted to 2 mg/mL with ACN. The IS stock solution was prepared by dissolving EPH8 in ACN at a concentration of 100 μg/mL. Stock solutions were stable for 6 months at −20°C.
A series of working solutions of JIB-04 were prepared by diluting stock solution with blank rat plasma to the appropriate concentrations (10 times as that of nominal concentration): 5, 25, 50, 250, 1000, 5000 and 10000 ng/mL. The working solutions for LLOQ and QC samples were independently prepared by spiking JIB-04 standard solutions with blank rat plasma to give the concentrations of 5, 10, 500, and 8000 ng/mL, which were ten times as that of nominal concentrations: 0.5 (LLOQ), 1 (LQC), 50 (MQC) and 800 ng/mL (HQC).
2.4. Sample processing
Eighteen microliters of blank rat plasma, 2 μL of working solution (or 20 μL of rat plasma for pharmacokinetic plasma samples) and 80 μL of ACN containing 5 ng/mL IS and 0.1% acetic acid were mixed by vortexing for 10 s, and then centrifuged for 20 min at 4 °C. The supernatant was transferred to a sample vial, and a 5 μL aliquot was injected for LC-MS/MS analysis.
2.5. Method validation
The analytical method was validated according to the FDA and the EMA guidelines for bioanalytical method validation [29, 30].
Selectivity was assessed by comparing the chromatograms of six different batches of blank plasma with the corresponding spiked plasma.
A standard curve in the form of y = Ax + B was determined by plotting the peak area ratio of JIB-04 to IS against known standard concentrations of JIB-04. The slope, intercept, and coefficient of determination were estimated using least squares linear regression method with a weighting of 1/x.
The triplicate injections of blank samples were conducted following six consecutive injections of upper limit of quantification (ULOQ=1000 ng/mL) QC samples. The peak area of the analyte for blank sample should be ≤20% of that for the LLOQ samples, the peak area of IS should be ≤5% of that of IS throughout the runs.
The Intra-day accuracy and precision were determined by replicate analyses (n = 6) of LLOQ (0.5 ng/mL) and QC samples (1, 50 and 800 ng/mL) on different validation days. Inter-day accuracy and precision were determined by six replicates of LLOQ and QC samples on three consecutive days. The relative error (RE%) was used to estimate the accuracy and coefficient of variation (CV%) was used to estimate the precision. The calculated results of inter-day and intra-day precision and accuracy should be ≤15% for QC levels and ≤20% for LLOQ.
The matrix effect and extraction recovery of JIB-04 were determined at four levels (LLOQ, LQC, MQC, and HQC) in six replicates. Matrix effects were evaluated by comparing the peak area ratio of the post-extracted QC samples (by spiking analytes in the extracted analyte-free blank plasma samples) to the peak area ratio of neat solution. Recoveries were calculated by comparing the mean peak area of spiked QC samples to those of post-extracted QC samples.
The stability of analytes was examined by keeping six replicates of the QC and LLOQ samples on the bench top at room temperature for 18 h, in the auto-sampler tray at 4°C for 24 h, and in a freezer at −80°C for 3 months; the freeze-thaw stability was obtained over three freeze-thaw cycles, by thawing at room temperature for 2–3 h and then refreezing at −80°C for 12–24 h. The concentration of analytes after each storage period was determined by using the calibration curve analyzed on the same day. The accuracy was expressed by RE% and the precision was CV%.
The dilution integrity experiment was required if the sample concentration was higher than the upper limit of quantitation (ULOQ). Dilution integrity was confirmed by measuring the accuracy and precision of 6 replicates of 10-fold HQC samples (8000 ng/mL) with a 10-fold dilution.
2.6. Animal study
The validated method was applied to investigate the plasma profiles of JIB-04 in rats after i.v. and oral administration of JIB-04. The animal study protocol (#9122) was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Texas Southern University.
Male adult SD rats were purchased from Envigo RMS (Indianapolis, IN, USA). Five rats were taken jugular vein cannulation and intravenously administered with a single dose of 5 mg/kg JIB-04 (12.5% cremophor EL, 12.5% DMSO, aqueous suspension prepared as previously reported [24]) via the jugular vein. Serial blood samples (about 0.2 mL) were collected from the cannulated jugular vein into heparin-coating tubes before dosing and at 2 min, 5 min, 15 min, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h, and 24.0 h time points after administration. Six rats were taken jugular vein cannulation and orally administered with a single dose of 15 mg/kg JIB-04. Serial blood samples were collected from the cannulated jugular vein into heparin-coating tubes before dosing and at 5 min, 15 min, 0.5 h, 1.0 h, 1.5 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h, and 24.0 h time points after administration. Plasma was separated immediately by centrifugation of the blood samples at 4000 rpm for 15 min and kept at −80°C until analysis.
Microsoft Excel 2016 software was used to process data analysis. Results were expressed as mean values with standard deviation. The pharmacokinetic parameters for each rat were estimated using Phoenix WinNonlin v7.0 software (Pharsight Corporation, Mountain View, CA, USA). Non-compartmental analysis was used to determine the pharmacokinetic parameters of JIB-04. The pharmacokinetic parameters including the maximum concentration (Cmax), the time that Cmax is reached (Tmax), the area under the plasma concentration–time curve during the period of observation (AUC0−24), the area under the plasma concentration–time curve extrapolated to infinity (AUC0−inf), apparent volume of distribution (Vd), apparent clearance (CL), total body mean residence time (MRT) and terminal elimination half-life were estimated. The oral bioavailability (F) was calculated according to the following equation:
3. Results and discussion
3.1. Method development
The Z- isomer was produced as the byproduct of the E-isomer during the synthesis of JIB-04 [18]. Because the Z-isomer is an inactive isomer, it was not as a target analyte in this study. However, to accurately quantify the E-isomer, we optimized the separation between the Z-isomer and the analyte E-isomer. The IS EPH8 is an analog of JIB-04, and their molecular weights are only 1 Da difference, so JIB-04 responded to the same transition for IS detection. Therefore, the two isomers of JIB-04 and IS should be well separated by retention time. Different columns, mobile phases and gradient programs were tested for the separation of JIB-04 and IS. ACE Excel 2 SuperC18 Column with water (0.05% acetic acid) as mobile phase A and ACN (0.05% acetic acid) as mobile B was selected because of the better signal response, peak shape and separation of E-isomer (retention time 1.43 min), Z- isomer (retention time 2.29 min), and IS (retention time 1.77 min) (Fig. 2).
Fig. 2.
Representative MRM chromatograms: (A) double blank rat plasma; (B) blank rat plasma spiked with JIB-04 (1 ng/mL) and IS (5 ng/mL); (C) a rat plasma sample at 2 h after i.v. administration of JIB-04 at a single dose of 5 mg/kg; (D) a rat plasma sample at 2 h after oral administration of JIB-04 at a single dose of 15 mg/kg.
Non-specific binding to plastics generally happens for proteins, and hydrophilic insoluble compounds can potentially non-specifically bind to plastic-ware as well [31]. For the proteins that show more affinity than the hydrophilic insoluble compounds, when the compounds in plasma are quantified, the non-specific binding is imperceptible; while the compounds are in neat aqueous solution, it may not be negligible. During the method development, JIB-04 was found strongly binding to plastic centrifuge tubes, which resulted in a limit of detection (LOD) of 2.5 ng/mL for JIB-04 in neat solution using water as the matrix and ACN as working solution solvent. To eliminate the non-specific binding, blank rat plasma and BSA solution were tested for the preparation of working solutions, and blank rat plasma showed better and stable response. In addition, water, 50% ACN and ACN were tested as the matrix to prepare neat solution and post-extraction QC samples, and 50% ACN achieved the optimal response.
The LOD of JIB-04 was as low as 0.05 ng/mL in rat plasma using a 6500+ Triple Quad LC-MS/MS System. In this study, JIB concentrations were between 2–6000 ng/mL in pharmacokinetic samples, we picked a reasonable LLOQ of 0.5 ng/mL with a linear range of 0.5–1000 ng/mL to quantify the samples. For other studies, the method could be validated at a lower LLOQ (such as 0.1 ng/mL).
3.2. Method validation
The method specificity was assured by analyzing six individual blank plasma samples, and there was no interference at the retention times of the analyte and IS. The selectivity was confirmed by analyzing six individual blank plasma fortified at the concentration of the LLOQ. The back-calculated values for these samples were within ± 20.0% of their theoretical value. Chromatograms of a blank rat plasma sample, a blank rat plasma sample spiked with JIB-04, and rat plasma samples at 2 h after i.v. and oral administration of JIB-04 are shown in Fig. 2. There was no carryover for both IS (≤5% of average response) and JIB-04 (≤20% of LLOQ).
The linearity of the calibration curves was found in the range of 0.5 – 1000 ng/mL. The linear regression correlation coefficients were greater than 0.999 in all validation runs.
The accuracy and precision were determined by replicate analyses (n = 6) of LLOQ (0.5 ng/mL) and QC (1, 50 and 800 ng/mL) samples on different validation days. Table 2 summarizes the precision and accuracy results. The intra-day accuracy (RE%) was −7.4% to 3.7%, and the precision (CV%) ranged from 1.9% to 7.6%; the inter-day accuracy ranged from −7.3% to 1.9%, and inter-day precision was between 3.8% to 10.2%. The precision and accuracy were within acceptance range according to FDA and EMA guidance. These data suggest that the method is accurate and precise for the quantification of JIB-04 in rat plasma.
Table 2.
Intra-day and inter-day accuracy and precision of JIB-04 in rat plasma.
| Nominal concentration (ng/mL) | Intra-day (n=6) | Inter-day (n=18) | ||||
|---|---|---|---|---|---|---|
| Observed concentration (mean ± SD) | Accuracy (RE%) | Precision (CV%) | Observed concentration (mean ± sD) | Accuracy (RE%) | Precision (CV%) | |
| 0.5 | 0.49 ± 0.03 | −2.1 | 7.1 | 0.51 ± 0.04 | 1.9 | 8.0 |
| 1 | 1.0 ± 0.1 | 3.4 | 7.6 | 0.93 ± 0.10 | −6.8 | 10.2 |
| 50 | 51.8 ± 3.0 | 3.7 | 5.8 | 49.9 ± 4.3 | −0.2 | 8.6 |
| 800 | 741.2 ± 14.2 | −7.4 | 1.9 | 741.9 ± 28.1 | −7.3 | 3.8 |
Matrix effects were investigated using the post-extraction spike method and ranged from 87.6 ± 1.8% to 101.0 ± 10.5% (Table 3), suggesting the matrix effect was negligible in this study. Recoveries were calculated by comparing the peak areas in QC samples to those of post-extracted QC samples. The recoveries were between 104.8 and 135.1% (Table 3). In QC samples, the analyte spiked in rat plasma, and the abundant proteins in plasma would relieve JIB-04 from non-specific binding to plastic tubes. At lower concentration, the relief of JIB-04 showed more significant effect on the recoveries, whereas at the higher concentrations, the effect was less.
Table 3.
Recovery and matrix effect of JIB-04 and IS.
| compounds | Nominal concentration (ng/mL) | Matrix effect (%) (n=6) | Recovery (%) (n=6) |
|---|---|---|---|
| JIB-04 | 0.5 | 92.5 ± 8.4 | 134.4 ± 8.1 |
| 1 | 87.6 ± 1.8 | 135.1 ± 2.7 | |
| 50 | 92.6 ± 0.8 | 121.4 ± 3.1 | |
| 800 | 101.0 ± 10.5 | 104.8 ± 1.5 | |
| IS (EPH8) | 5 | 68.3 ± 4.9* | 100.6 ± 5.3* |
n=24
The stability of JIB-04 was evaluated as described in the methods section. Data shown in Table 4 suggest that no significant degradation occurred under the experimental conditions. The plasma samples could be stored at −80 °C for as long as 3 months without apparent alteration.
Table 4.
Stability data for JIB-04 in rat plasma
| Stability test | Nominal Concentration (ng/mL) | Calculated Concentration (ng/mL) | ||
|---|---|---|---|---|
| Mean ± SD (n=6) | RE (%) | CV (%) | ||
| auto-sampler (10 °C 24hr) | 0.5 | 0.48 ± 0.01 | −3.2 | 2.8 |
| 1 | 0.97 ± 0.06 | −3.5 | 5.8 | |
| 50 | 48.6 ± 3.3 | −2.7 | 6.8 | |
| 800 | 799.0 ± 17.5 | −0.1 | 2.2 | |
| bench top (18hr, RT) | 0.5 | 0.51 ± 0.02 | 1.6 | 4.0 |
| 1 | 0.93 ± 0.05 | −6.9 | 5.6 | |
| 50 | 46.8 ± 0.7 | −6.4 | 1.6 | |
| 800 | 819.0 ± 26.0 | 2.4 | 3.2 | |
| Freeze and thaw (−80 °C to RT) | 0.5 | 0.51 ± 0.01 | 1.4 | 2.9 |
| 1 | 0.99 ± 0.03 | −0.8 | 3.2 | |
| 50 | 49.0± 1.3 | −2.1 | 2.6 | |
| 800 | 793.0 ± 16.2 | −0.9 | 2.0 | |
| Long-term (−80 °C, 3 months) | 0.5 | 0.47 ± 0.02 | −6.7 | 3.5 |
| 1 | 0.93 ± 0.05 | −6.9 | 4.9 | |
| 50 | 47.2 ± 1.3 | −5.6 | 2.8 | |
| 800 | 745.0 ± 20.8 | −6.9 | 2.8 | |
Dilution integrity ensures that dilution of a specimen with a concentration higher than ULOQ could result in an accurate quantification. Six 10-fold HQC samples were diluted 10 times and analyzed. The accuracy was 0.8% and the precision was 4.5%, which suggests that proper dilution is acceptable for the method.
3.3. Pharmacokinetic study
The validated method was applied to investigate the plasma profiles of JIB-04 in rats after i.v. and oral administration of JIB-04. In the plasma samples after i.v. and oral administration, E-isomer of JIB-04 was detectable right after dosing (2 min for i.v. administration, 5 min for oral administration) until 24 h post administration. Z-isomer was also observable at the same period. The E/Z ratios (72 at 5 min, 27 at 2 h, and 0.6 at 24 h; in oral samples in i.v. samples; 40 at 5 min, 9 at 2 h, and 2.3 at 24 h in oral samples) decreased along with time change, which suggested that a fraction of E-isomer was converted to Z-isomer in vivo. The metabolic mechanism needs further investigation.
Mean plasma concentration vs. time profiles are presented in Fig. 3. The main pharmacokinetic parameters calculated using Phoenix Winnonlin software by noncompartmental analysis are listed in Table 5.
Fig. 3.
Mean plasma concentration–time profile of JIB-04 in rat plasma after intravenous (n=5) and oral (n=6) administration.
Table 5.
Pharmacokinetic parameters of JIB-04 after i.v. and oral administration of JIB-04 to rats (data derived from NCA)
| Parameters | MEAN ± SD | |
|---|---|---|
| i.v. (5 mg/kg) (n=5) | oral (15 mg/kg) (n=6) | |
| Cmax (ng/mL) | − | 812.7 ± 201.2 |
| Tmax (h) | − | 1.5 ± 1.4 |
| AUC 0→24 (h·ng/mL) | 3661.6 ± 371.4 | 4880.8 ± 1015.3 |
| AUC 0→inf (h·ng/mL) | 3678.2 ± 369.0 | 4904.7 ± 1019.7 |
| Half-life (h) | 3.70 ± 0.5 | 3.2 ± 0.2 |
| CL (mL/h/kg) | 1370.7 ± 141.7 | 3234.2 ± 76.3 |
| Vd (ml/kg) | 7357.6 ± 1530.6 | 14923.4 ± 3669.9 |
| MRT (h) | 2.6 ± 0.3 | 4.4 ± 0.3 |
-: not applicable
Following i.v. administration, high JIB-04 concentrations (5270 ± 527.2 ng/mL at 2 min) in plasma was observed immediately after dosing followed by a rapid decrease. After 24 h, JIB-04 concentration was only 3.0 ± 1.0 ng/mL. Following oral administration, JIB-04 was quickly absorbed into blood. JIB-04 concentration reached 122.9 ± 51.0 ng/mL within 5 min, and reached the Cmax in 0.5 ~ 4 h, then rapidly decreased. Four out of six subjects showed a 2nd peak concentration, and two subjects only showed one peak concentration, which implies there is individual difference among subjects. The oral bioavailability of JIB-04 was calculated to be 44.4%.
4. Conclusion
JIB-04 is a small molecule that pan-selectively inhibits the demethylase activity of KDMs in vitro, in cancer cells, and in vivo; it blocks HIV-1 Tat expression, transactivation, and virus replication via the inhibition of SHMT2 in T cell lines. In the present study, a sensitive, specific, fast, and reliable LC- MS/MS method for the quantification of JIB-04 in rat plasma samples was developed and fully validated according to FDA and EMA guidelines. The calibration curves were linear over concentrations ranging from 0.5 to 1000 ng/mL. The accuracy (RE%) was between −7.4% and 3.7%, and the precision (CV%) was 10.2% or less. The method has been successfully applied to pharmacokinetic studies for the quantification of JIB-04 after i.v. and oral administration in rats.
Highlights.
JIB-04 has brought outstanding interest as a pan-selective inhibitor of KDMs.
A UHPLC-MS/MS method was validated for the quantification of JIB-04 in rat plasma.
This assay was applied to the pharmacokinetic study of JIB-04 in rat plasma.
Acknowledgements
This study was funded in part by Cancer Prevention & Research Institute of Texas (CPRIT) Core Facilities Support Awards (RP180748), and the National Institute of Health’s Research Centers in Minority Institutes Program (RCMI, G12MD007605) to H.X. and D.L.; as well as by the Welch Foundation (I-1878), and by the John P. Perkins, Ph.D Endowment in Biomedical Science to E.D.M. We acknowledge Dr. Jung-Mo Ahn’s lab at University of Texas at Dallas for synthesizing and providing JIB-04.
Footnotes
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Declaration of competing interest
The authors declare no conflicts of interest.
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