1. Introduction
The blockade of the adenosine A
2A receptor in striatopallidal neurons is known to reduce the postsynaptic effect of dopamine depletion and the motor deficit of Parkinson’s disease (PD). Because of these properties, adenosine A
2A receptor antagonists are well known as good target drug candidates for the treatment of PD [
1,
2,
3,
4,
5,
6,
7].
Recently, as interest in immunotherapy has increased, many articles and papers have shown that adenosine pathways are also involved in immunosuppression [
8,
9,
10]. The pathway of adenosine production by 5′-nucleotidase is one of the immunosuppressive pathways [
8,
9]. The expression of 5′-nucleotidase enhances tumor progression and metastasis [
8,
9,
11,
12]. CD39/CD73 expressed in tumors convert Adenosine triphosphate into adenosine [
8,
9,
10,
12,
13,
14]. This adenosine binds to the adenosine A
2A receptor to increase cAMP, and this cAMP inhibits immune response factors such as natural killer cells, dendritic cells, and T cells [
10,
11,
13,
15]. This chain reaction may put the brakes on the anti-tumor immune response against the tumor, while it enhances tumor progression and metastasis [
10,
11,
13,
14]. Conversely, when the adenosine A
2A receptor antagonist is applied to inhibit the receptor, an immune response may occur, thus exhibiting an anti-tumor effect [
8,
10,
11,
13,
14,
15].
5-Amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c) pyrimidine (SCH 58261) is classified as an adenosine A
2A receptor antagonist [
6,
7,
16,
17,
18,
19] (
Figure 1). The non-xanthine heterocyclic compound SCH 58261 is a new, potent and selective A
2A adenosine receptor antagonist [
18]. This drug particularly binds to the A
2A receptor, among the various adenosine receptor subtypes [
18,
19]. It is known that SCH 58261 is potent against cAMP production as well as binding [
17,
18,
20]. SCH 58261 is one of the new chemical entities that has been developed as an adenosine A
2A receptor antagonist. Since then, SCH 58261 has been widely used as a reference drug in the development of adenosine A
2A receptor antagonists in many articles [
21,
22,
23,
24,
25]. However, although SCH 58261 has been reported to be beneficial, there is little information about SCH 58261 in terms of its absorption, distribution, metabolism, and excretion (ADME) as well as its pharmacokinetics (PK) perspectives in animals and humans.
The response of a drug in in vivo animal models conventionally depends on its absorption, distribution, metabolism, and elimination associated with the pharmacokinetic factors [
26]. Therefore, the ADME knowledge in the chemical series of drugs is essential for successful structural optimization and design during lead optimization and the candidate selection process. Bioavailability quantifies the proportion of a drug that is absorbed and available to produce systemic effects [
27,
28]. Oral bioavailability is one of the key factors frequently assessed in drug development. In general, the factors affecting drug bioavailability after oral administration include (but are not limited to) absorption and metabolism [
26].
The purpose of this study was to explore the metabolism and pharmacokinetic properties of SCH 58261 to understand its behaviors in vivo. Male Sprague–Dawley (SD) rats (8 weeks old) were used as an in vivo model species. First, an LC–MS/MS method was developed for the determination of SCH 58261 in rat plasma. GastroPlus™ is a tool to simulate the PK properties of drugs in virtual humans and animals [
29]. Parameter sensitivity analysis is one of the built-in modules of GastroPlus™, which is useful for understanding the sensitivities in PK profiles between various PK parameters [
30,
31,
32]. In vitro microsomal metabolic stability, in vitro metabolite identification, and bile duct-cannulated studies were also conducted to understand the PK characteristics of SCH 58261. In vitro microsomal metabolic stability can be determined by in vitro half-life (t
1/2) and intrinsic clearance (CL
int) using a simple in vitro experiment [
33,
34,
35]. This value can be used to extrapolate in vivo intrinsic clearance and hepatic clearance [
33,
35]. Since the liver is considered to be one of the main organs involved in drug metabolism, the hepatic clearance value obtained from the in vitro study would make it possible to predict how much metabolism could occur in the liver [
33,
35]. The identification of metabolites using rat liver microsomes is an experiment that can easily predict metabolites generated by the liver metabolic enzymes [
36]. The bile duct-cannulated rat study is a test to see the amount of drug excreted in each sample after collecting the feces, urine, and bile, which is obtained by connecting the tube to the bile duct of the rat [
37]. Overall, the following factors such as permeability, systemic clearance, renal clearance, and liver first-pass effect, etc., will be explored from various in vitro and in vivo experiments in this study to understand the drug metabolism and pharmacokinetic properties of SCH 58261.
3. Materials and Methods
3.1. Materials
SCH 58261 was purchased from MedChem Express (Monmouth Junction, NJ, USA). Uridine 5’-diphosphoglucuronic acid triammonium salt (UDPGA), nicotinamide adenine dinucleotide phosphate reduced (NADPH), glutathione (GSH) and verapamil, which was used for the internal standard (ISTD), were purchased from Sigma-Aldrich (St Louis, MO, USA). Male SD rat liver microsomes were purchased from Corning Incorporated (Corning, NY, USA). Acetonitrile (ACN) of HPLC grade was purchased from Honeywell Burdick & Jackson (Ulsan, Korea). Distilled water (DW) of HPLC grade was purchased from Samchun Chemical (Gyeonggi-do, Korea). All other chemicals and solvents were commercial products of analytical or reagent grade and were used without further purification.
3.2. Stock Solution
The stock solution of SCH 58261 was prepared by dissolving the drug in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL and storing it in a refrigerator at −20 °C until use. The 0.1 mg/mL sub-stock solution was made by spiking 100 µL of stock solution in 900 µL of DMSO. ISTD was prepared by dissolving verapamil in ACN at a concentration of 100 ng/mL.
3.3. Preparation of Calibration Curve STD and QC Samples
The sub-stock solution of SCH 58261 was further diluted with DMSO to give a series of calibration curve standards, with concentrations ranging from 3.02 to 2200 ng/mL. The calibration curve standard samples were prepared in duplicate and the standard curves were obtained by establishing a quadratic regression function, with an equation y = ax2 + bx + c after 1/concentration2 weighting. QC samples with concentrations of 15.02 (QC low), 165.46 (QC medium) and 1820 (QC high) ng/mL were prepared in the same way as the calibration curve STDs.
3.4. Sample Preparation and Extraction Procedures
For each 20 µL of blank rat plasma, 4 µL of the STD or QC working solutions were added and vortexed for 1 min to mix. On the other hand, for blank samples, 4 µL of make-up solution (DMSO) was added to blank rat plasma. For study samples, 4 µL of make-up solution (DMSO) was also added to rat PK study samples to assure the same matrix conditions as STD and QC samples. Then, 100 µL of ACN containing 100 ng/mL verapamil as ISTD was added to the mixture for protein precipitation. After vortexing for 1 min and centrifuging at 10,000 rpm for 5 min, the supernatant was transferred to another Eppendorf tube. Finally, the samples were diluted three times with DW and the mixture was transferred to an LC vial for LC–MS/MS analysis.
3.5. Equipments and Chromatographic Conditions
The liquid chromatography–mass spectrometry system is composed of a Shimadzu CBM-20A HPLC pump controller (Shimadzu Corporation, Columbia, MD, USA), two Shimadzu LC-20AD pumps, a CTC HTS PAL auto-sampler (LEAP Technologies, Carrboro, NC, USA) and a Triple time-of-flight (TripleTOF
®) 5600 mass spectrometer (Sciex, Foster City, CA, USA) with an electrospray ionization (ESI) source. The chromatographic separation was achieved on a Phenomenex Kinetex XB-C18 column (2.1 × 100 mm for metabolite identification and 2.1 × 50 mm for method qualification), and the column temperature was set to 50 °C. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile, with a binary flow rate of 0.4 mL/min and an injection volume of 10 μL. The LC gradients for SCH 58261 quantification and metabolite identification are summarized in
Table 9.
The TripleTOF® 5600 mass spectrometer was equipped with a TurboIonSpray® ion source Electrospray ionization (Sciex, Redwood City, CA, USA) was used for sample introduction and ionization in the positive ion mode. High-purity nitrogen gas was used for the nebulizer/Duospray™ and curtain gases. Source-dependent parameters optimized were as follows: GS1 and GS2—both 50 psi; ion spray voltage—5500 V; temperature—500 °C, with a curtain gas flow of 30 L/min. The compound-dependent parameters such as the declustering potential (DP) and collision energy (CE) were optimized during tuning as 160 V and 35 V for SCH 58261, and 125 V and 30 V for verapamil, respectively. Based on the full scan and MS/MS spectrum, the transitions of precursors to the product ions were as follows: m/z 346.1 → 105.1 for SCH 58261 and m/z 455.2 → 165.2 for Verapamil. Data acquisition and analysis were performed using the Analyst® TF 1.6 software (Sciex, Foster City, CA, USA).
3.6. Method Qualification
The current LC–MS/MS assay was qualified in respect of linearity, intra-day and inter-day accuracy and precision and stability. The calibration curve was acquired by plotting the ratio between the peak area of SCH 58261 and the internal standard against the nominal concentration of calibration standards. The final concentrations of calibration standards obtained to plot the calibration curve were 3.02, 9.05, 27.16, 81.48, 244.44, 733.33, and 2200 ng/mL. The calibration curves were fitted by a weighted (1/concentration2) quadratic regression function, with an equation y = ax2 + bx + c. The criteria for the acceptability of the data were within ±25% accuracy and precision, except for the LLOQ, where it should not exceed ±30% accuracy, as well as precision. In addition, each run contained blank plasma samples with and without internal standards in duplicate.
Precision and accuracy were evaluated by determining the SCH 58261 concentrations in three replicates of QC samples freshly prepared in a daily base at three different concentrations for three separate days.
The dilution integrity experiment was carried out at five times the QC high concentration, i.e., 9100 ng/mL. Three replicate samples were prepared and their concentrations were calculated by applying the dilution factor of five against the freshly prepared calibration curve for SCH 58261. The acceptance criteria for dilution integrity were within ±25% precision and accuracy.
Stability assessments were performed to evaluate the SCH 58261 stability in stock solutions and plasma samples during study sample handling and analysis under different conditions. Stability assessments were performed by comparing the peak area ratio (SCH 58261 peak area/ISTD peak area) of the study samples against the freshly prepared control samples. The stability evaluations were conducted using triplicates of QC samples in four stability tests such as stock solution, short-term, long-term and freeze–thaw stability; stock solution stability after storage at −20 °C for 28 days; short-term stability after storage at room temperature for 4 h; long-term stability after storage at −80 °C for 28 days; freeze–thaw stability through three freeze–thaw cycles at −80 °C to 25 °C. Samples were considered to be stable if assay values were within ±25% accuracy and precision.
For species-dependent matrix effect tests, triplicate QC samples were prepared in mouse, dog, and human plasma. Samples were quantitated with a calibration curve prepared in rat plasma. The acceptance criterion was within ±25% of precision and accuracy.
3.7. Pharmacokinetic Study in Rats
All animal studies were performed in accordance with the “Guidelines in Use of Animal” established by the Chungnam National University Institutional Animal Care and Use Committee (Daejeon, Korea). This study was approved by the Chungnam National University Institutional Animal Care and Use Committee (No. CNU-01104). Male SD rats (8 weeks old; Samtako Biokorea, Gyeong-gi, Korea), 280–300 g, were used for the pharmacokinetic study. For the PK study, two groups of cannulated rats were administered a single 1 mg/kg intravenous dose and a single 5 mg/kg oral dose of SCH 58261. Blood samples were collected at 2, 5, 15, 30, 60, 90, 120, and 240 min following drug administration in the intravenous group (n = 3). Blood samples were collected 5, 30, 90 and 240 min for oral doses (n = 2). After centrifugation at 10,000 rpm for 5 min, plasma was transferred to Eppendorf tubes and stored at −20 °C until analysis. After sample preparation, the PK samples were analyzed by LC–MS/MS.
Pharmacokinetic parameters were obtained on each individual set of data using the Phoenix WinNonlin™ software (version 8.0; Pharsight Corporation, Mountain View, CA, USA) by non-compartmental analysis (NCA).
3.8. Parameter Sensitivity Analysis Using the Compartment Model
The compartment model of GastroPlus™ (version 9.5; Simulations Plus, Inc., Lancaster, CA, USA) was used for parameter sensitivity analysis. When simulating one-, two- and three-compartment models based on SCH 58261 structure, the most fitted model with the drug concentration that was observed after intravenous administration was selected. The selected model was a two-compartment model. The factors that influence the exposure of the drug after oral administration using the two-compartment model were examined through parameter sensitivity analysis. The drug exposure was assessed as AUClast. The total of seven factors were simulated for drug exposure: permeability, solubility, systemic clearance, renal clearance, FPE, blood–plasma ratio, and unbound fraction.
3.9. Microsomal Metabolic Stability
Rat liver microsomes (final concentration = 1 mg/mL) were incubated with SCH 58261 (1.5 µg/mL) in a reaction mixture that consisted of 2 mM NADPH and 5 mM UDPGA at 37 °C. All incubations were performed in triplicate and the reaction was initiated by adding the cofactor solutions containing NADPH and UDPGA to the rat liver microsome suspension with a 3-min pre-incubation. The microsomes, mixed-cofactor, and SCH 58261 were mixed and incubated for 0, 15, 30, and 60 min and the reaction was stopped by adding 50% ACN/50% methanol (MeOH) containing 100 ng/mL verapamil. After vortexing for 1 min and centrifuging at 8000× g for 10 min, the supernatant was transferred to another Eppendorf tube. Finally, the samples were diluted three times with DW and the mixture was transferred to an LC vial for LC–MS/MS analysis.
3.10. In Vitro Metabolite Identification
Rat liver microsomes (final concentration = 2 mg/mL) were incubated with SCH 58261 (5 µg/mL) in a reaction mixture that consisted of 2 mM NADPH, 5 mM UDPGA, and 0.5mM GSH at 37 °C. The reaction was initiated by adding the cofactor solutions containing NADPH, UDPGA, and GSH to the rat liver microsome suspension with a 3-min pre-incubation. The microsomes, mixed-cofactor, and SCH 58261 were mixed and incubated for 0 and 60 min at 37 °C. The reaction was stopped by adding ACN. After vortexing for 1 min and centrifuging at 8000× g for 10 min, the supernatant was transferred to another Eppendorf tube. The supernatants were evaporated to dryness under vacuum in a rotary evaporator with a cold trap (Eyela CVE-3110 & UT-1000, Tokyo, Japan). The dried residue was re-constituted to 210 μL of DW/MeOH (2:1), vortexed, centrifuged at 10,000× g for 5 min, and the supernatant was transferred to an LC vial for analysis. PeakView® Version 2.2 (Sciex, Redwood City, CA, USA) and MetabolitePilot™ Version 2.0.2 (Sciex, Redwood City, CA, USA) were used for the structural elucidation of SCH 58261 metabolites.
3.11. Bile Duct-Cannulated Rat Study
A bile duct-cannulated rat study was conducted to evaluate the elimination pathway of SCH 58261. Samples of urine, bile, and feces were collected for 24 hours after a single 5 mg/kg oral dose (n = 1) from rat metabolic cages (JeungDo Bio & Plant co., Seoul, Korea). Urine samples were diluted twofold with 30% ACN. Feces samples were subsequently homogenized in four volumes of phosphate-buffered saline (PBS, fivefold dilution). The obtained samples were further processed and analyzed using LC–MS/MS.
4. Conclusions
SCH 58261 is one of the drugs being developed as an adenosine A2A receptor agonist and has been used in many papers as a reference drug in the development of an adenosine A2A receptor antagonist. However, there are no reports that clearly demonstrate the ADME/PK aspect of SCH 58261. Therefore, we investigated the ADME/PK of SCH 58261 as a reference drug for the development of adenosine receptor antagonists.
First, the LC–MS/MS method was established to accurately quantify SCH 58261 in rat plasma. The calibration curve range is 3.02 to 2200 ng/mL. This range was sufficient enough to cover the concentration of the drug when administered at 1 mg/kg intravenously and 5 mg/kg orally in rats. The correlation coefficient of the calibration curve showed a linearity of ≥0.99. The LC–MS/MS method has also been shown to be sensitive, selective, accurate, and reproducible through a variety of stability experiments (stock, short-term stability, long-term stability, and freeze–thaw stability) and achieved dilution integrity. Therefore, this method has been applied successfully to various in vitro and in vivo PK studies.
Then, in vivo PK studies for SCH 58261 were carried out in rats. The SCH 582631 was administered intravenously at 1 mg/kg and orally at 5 mg/kg, and the drug concentration in rat plasma was monitored. These studies showed that the bioavailability (BA) of SCH 58261 was extremely low when given orally and the root cause of the low BA was investigated.
A simple in silico method was used to find the root cause of the poor BA of SCH 58261 when given orally. The GastroPlus™ was used as an in silico tool. The parameter sensitivity analysis of GastroPlus™ examined the factors that influence the exposure of SCH 58261, such as AUClast. The factors that had a great effect on AUClast for oral administration were permeability, systemic clearance, renal clearance, and liver FPE%. Therefore, we hypothesized that one (or a number) of these factors would have a significant effect on the low BA of SCH 58261.
Microsomal metabolic stability was performed to evaluate the metabolism of SCH 58261 in rat liver microsomes. The hepatic clearance value obtained from this experiment was 39.97 mL/min/kg. Assuming a rat liver blood flow of 55 mL/min/kg, the hepatic clearance value obtained was as high as 72% of the rat liver blood flow. Therefore, SCH 58261 is a drug that is highly metabolized by the liver, and it was supposed that one of the reasons for poor oral BA might be due to significant hepatic metabolism.
In vitro metabolite identification shows that the metabolite type of SCH 58261 in the rat liver is mono-oxidation or ketone-forming metabolites. A total of four metabolites were identified, three mono-oxidized metabolites and one metabolite-formed ketone. No significant metabolites greater than SCH 58261 appeared to be present, with an assumption of similar ionization efficiency between SCH 58261 and its metabolites.
Finally, a bile duct-cannulated rat study was conducted. Bile, urine, and feces samples from rats were collected to identify the main excretion route for SCH 58261 after oral administration. Approximately 28.17% of SCH 58261 administered was excreted in the first 24 h without absorption. This information implies that a major contribution to the extremely low BA of SCH 58261 would be likely due to no/little GI absorption after oral administration. Even if a small fraction of SCH 58261 was absorbed, SCH 58261 would be subject to significant drug metabolism by CYPs metabolic enzymes in the liver, which further contributed to the low BA of SCH 58261 in vivo. In conclusion, the low BA of SCH 58261 after oral administration in rats would be due to a limited absorption process (primary reason) with a high metabolism (secondary reason) in vivo.
In the future, researchers who are developing new SCH58261 analogs should be aware of the relationship between drug exposure and biomarkers in order to gain a better understanding of their pharmacokinetics/pharmacodynamics(PK/PD) for efficacy; otherwise, it would be challenging to predict efficacious doses in preclinical and clinical studies of the poor oral exposure of drugs. Several novel strategies to overcome this poor oral drug exposure, such as new formulations or pro-drug approaches, would warrant exploration for this purpose based on the PK and physicochemical properties of SCH58261 analogs.