In Vitro Metabolic Pathways of the New Anti-Diabetic Drug Evogliptin in Human Liver Preparations

Evogliptin ((R)-4-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(tert-butoxymethyl)-piperazin-2-one), is a new dipeptidyl peptidase IV inhibitor used for the treatment of type II diabetes mellitus. The in vitro metabolic pathways of evogliptin were identified in human hepatocytes, liver microsomes, and liver S9 fractions using liquid chromatography-Orbitrap mass spectrometry (LC-HRMS). Five metabolites of evogliptin-4-oxoevogliptin (M1), 4(S)-hydroxyevogliptin (M2), 4(R)-hydroxyevogliptin (M3), 4(S)-hydroxyevogliptin glucuronide (M4), and evogliptin N-sulfate (M5)—were identified in human liver preparations by comparison with authentic standards. We characterized the cytochrome P450 (CYP) enzymes responsible for evogliptin hydroxylation to 4(S)-hydroxyevogliptin (M2) and 4(R)-hydroxyevogliptin (M3) and the UGT enzymes responsible for glucuronidation of 4(S)-hydroxyevogliptin (M2) to 4(S)-hydroxy-evogliptin glucuronide (M4). CYP3A4/5 played the major role in the hydroxylation of evogliptin to 4(S)-hydroxyevogliptin (M2) and 4(R)-hydroxyevogliptin (M3). Glucuronidation of 4(S)-hydroxy-evogliptin (M2) to 4(S)-hydroxyevogliptin glucuronide (M4) was catalyzed by the enzymes UGT2B4 and UGT2B7. These results suggest that the interindividual variability in the metabolism of evogliptin in humans is a result of the genetic polymorphism of the CYP and UGT enzymes responsible for evogliptin metabolism.


Introduction
Type II diabetes mellitus (DM) is a chronic metabolic disorder, characterized by relative insulin deficiency due to disorders of insulin secretion and insulin resistance, the prevalence of which has increased continually in the majority of countries [1]. Various classes of oral antidiabetic drugs can be used to control the blood glucose level and to prevent diabetic complications such as diabetic nephropathy and retinopathy [1,2].
Evogliptin (DA-1229, trade name: Sugarnon ® ), a new, potent, and selective DPP-IV inhibitor [6][7][8][9][10][11], was approved by the Ministry of Food and Drug Safety of Korea as an oral antihyperglycemic drug for the treatment of type II DM on October 2 2015. Although the pharmacokinetic properties of evogliptin in humans have been reported [9,10], there is no report of its in vitro metabolism in humans. Metabolite identification and characterization of drug-metabolizing enzymes-such as cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT)-responsible for the metabolism of drugs can facilitate prediction of interindividual variations in drug metabolism and pharmacokinetics, together with drug-drug interactions [12][13][14]. The purposes of the present study were to identify the metabolites of evogliptin formed after incubation with human hepatocytes, liver microsomes, and liver S9 fractions in the presence of cofactors using liquid chromatography-Orbitrap mass spectrometry (LC-HRMS), and to characterize the CYP and UGT enzymes responsible for evogliptin metabolism.

Identification of the Metabolites of Evogliptin in Human Hepatocytes
Cryopreserved human hepatocytes were purified and recovered using a cryohepatocyte purification kit (Woburn) according to the manufacturer's protocol. Purified human hepatocytes were resuspended in William's E medium to a final density of 1.28 × 10 6 cells/mL, and then 62.5 μL of human hepatocyte suspensions (8 × 10 4 cells) and 62.5 μL of 100 μM evogliptin in William's E medium were added to a 96-well plate and incubated for 4 h at 37 °C in a CO2 incubator. The reaction was terminated by the addition of 125 μL of ice-cold acetonitrile to each sample well, followed by centrifugation at 13,000 rpm for 4 min at 4 °C. Then, 40 μL of the supernatant were diluted with 60 μL of deionized water and an aliquot (5 μL) was injected onto LC-HRMS system to identify metabolites of evogliptin.

Identification of Metabolites of Evogliptin in Human Liver S9 Fractions
The incubation mixture consisted of 50 mM potassium phosphate buffer (pH 7.4), 10 mM magnesium chloride, pooled human liver S9 fractions (150 μg protein), 1 mM NADPH, 2 mM UDPGA, 0.2 mM PAPS and 10 μM evogliptin in a total volume of 300 μL. The incubation mixture was incubated for 60 min at 37 °C in a shaking water bath. The incubation was stopped by the addition of 300 μL of ice-cold acetonitrile, followed by centrifugation at 13,000 rpm for 4 min at 4 °C. The supernatant was evaporated using a vacuum concentrator and the residue was dissolved in 100 μL of 15% acetonitrile. An aliquot (5 μL) was injected onto the LC-HRMS system.

Identification of Metabolites of Evogliptin in Human Liver Microsomes
The incubation mixture consisted of 50 mM potassium phosphate buffer (pH 7.4), 10 mM magnesium chloride, pooled human liver microsomes (60 μg protein), 1 mM NADPH, 2 mM UDPGA, and 10 μM evogliptin in a total volume of 300 μL. The reaction mixture was incubated for 60 min at 37 °C in a shaking water bath. The incubation was stopped by the addition of 300 μL of ice-cold acetonitrile, followed by centrifugation at 13,000 rpm for 4 min at 4 °C, and the supernatant was evaporated. The residue was reconstituted in 100 μL of 15% acetonitrile, and an aliquot (5 μL) was analyzed by LC-HRMS.
The reaction mixture comprised 50 mM potassium phosphate buffer (pH 7.4), 10 mM magnesium chloride, pooled human liver microsomes (15 μg protein), and various concentrations of evogliptin (10 to 800 μM; final acetonitrile concentration not exceeding 0.5%, v/v) was preincubated for 3 min at 37 °C. The reaction was initiated by adding NADPH, and the mixture was further incubated (final volume of 100 μL) for 20 min at 37 °C in a shaking water bath. The reaction was terminated by adding 100 μL of MPPI (internal standard, 50 ng/mL) in ice-cold acetonitrile. The mixture was centrifuged at 13,000 rpm for 4 min at 4 °C. Subsequently, 50 μL of the supernatant were diluted with 50 μL of deionized water and an aliquot (5 μL) was injected onto the LC-MS/MS system.

Metabolism of Evogliptin in Human cDNA-Expressed CYP Enzymes
The reaction mixture comprised 50 mM potassium phosphate buffer (pH 7.4), 10 mM magnesium chloride, evogliptin (5 or 50 μM), and 12 human cDNA-expressed CYP enzymes (CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, and 3A5; 4 pmol) and was preincubated for 3 min at 37 °C. The reaction was initiated by addition of NADPH, and the mixture was further incubated (final volume of 100 μL) for 20 min at 37 °C in a shaking water bath. The reaction was terminated by adding 100 μL of MPPI (internal standard, 50 ng/mL) in ice-cold acetonitrile. The mixture was centrifuged at 13,000 rpm for 4 min at 4 °C. Continuously, 50 μL of the supernatant were diluted with 50 μL of deionized water and an aliquot (5 μL) was analyzed using the LC-MS/MS system.
For the enzyme kinetic experiments, various concentrations of evogliptin (10 to 800 μM; final acetonitrile concentration not exceeding 0.5%, v/v) were incubated with human cDNA-expressed CYP enzymes (CYPs 3A4 and 3A5; 4 pmol), 1 mM NADPH, and 10 mM MgCl2 in 50 mM potassium phosphate buffer (pH 7.4) for 20 min at 37 °C in a shaking water bath. After addition of ice-cold acetonitrile containing internal standard (MPPI, 50 ng/mL), the mixture was centrifuged and diluted as described above, and an aliquot (5 μL) was injected onto the LC-MS/MS system.

Correlation Analysis of Evogliptin Metabolism with Probe Substrate Activities in Human Liver Microsomes
Evogliptin (10 and 50 μM) was incubated with 10 different human liver microsomes (15 μg protein), 1 mM NADPH, and 10 mM magnesium chloride in 50 mM potassium phosphate buffer (pH 7.4) for 20 min at 37 °C in a shaking water bath. The correlation coefficients between the formation rates of 4(S)-hydroxyevogliptin (M2) or 4(R)-hydroxyevogliptin (M3) from evogliptin and specific CYP activities in human liver microsomes provided by Corning Life Sciences were evaluated by the Pearson product-moment correlation coefficient using Sigma Stat software (ver. 2.0; Systat Software Inc., San Jose, CA, USA). A p value < 0.05 was considered to indicate significance.

Immunoinhibition of Evogliptin Metabolism with an Anti-CYP3A4 Antibody
Immunoinhibition experiments were conducted by incubating pooled human liver microsomes with various amounts of an anti-CYP3A4 antibody for 15 min on ice, and then the reaction was initiated by the addition of 50 mM potassium phosphate buffer (pH 7.4), 50 μM evogliptin, 10 mM magnesium chloride, and 1 mM NADPH. Control incubations were performed using liver microsomes and 25 mM Tris buffer but without the anti-CYP3A4 antibody, which was prepared in this buffer.

Enzyme Kinetics for the Metabolism of 4(S)-Hydroxyevogliptin to 4(S)-Hydroxyevogliptin Glucuronide (M4) in Human Liver Microsomes and cDNA-Expressed UGT Enzymes
Preliminary experiments showed that the glucuronidation of 4(S)-hydroxyevogliptin to 4(S)-hydroxyevogliptin glucuronide (M4) was linear with incubation time over 30 min and human liver microsomal protein concentration (0.1-0.3 mg/mL). Therefore, a 20 min incubation time and 0.2 mg/mL microsomal protein concentration were selected for enzyme kinetics experiments. The reaction mixture comprised 50 mM Tris buffer (pH 7.4), 10 mM magnesium chloride, 0.025 mg/mL alamethicin, pooled human liver microsomes (20 μg protein), and various concentrations of 4(S)-hydroxyevogliptin (M2) (10 to 2,000 μM; final acetonitrile concentration not exceeding 0.5%, v/v), and was preincubated for 3 min at 37 °C. The reaction was initiated by adding UDPGA, and the mixture was further incubated (final volume of 100 μL) for 20 min at 37 °C in a shaking water bath. The reaction was stopped by adding 100 μL of MPPI (internal standard, 50 ng/mL) in ice-cold acetonitrile. The mixture was centrifuged at 13,000 rpm for 4 min at 4 °C. Next, 50 μL of the supernatant were diluted with 50 μL of deionized water and an aliquot (5 μL) was injected onto the LC-MS/MS system.

Correlation Analysis of 4(S)-Hydroxyevogliptin Metabolism to 4(S)-Hydroxyevogliptin Glucuronide (M4) with Probe Substrate Activities in Human Liver Microsomes
The formation rates of 4(S)-hydroxyevogliptin glucuronide (M4) from 4(S)-hydroxyevogliptin (M2) were evaluated by incubating 4(S)-hydroxyevogliptin (100 μM) with 10 different human liver microsomes (20 μg protein), 2 mM UDPGA, 0.025 mg/mL alamethicin, and 10 mM magnesium chloride in 50 mM Tris buffer (pH 7.4) for 20 min at 37 °C in a shaking water bath. The correlation coefficients between the formation rates of 4(S)-hydroxyevogliptin glucuronide (M4) and specific UGT activities in human liver microsomes reported by Corning Life Sciences were determined by the Pearson product-moment correlation coefficient using Sigma Stat Software (Systat Software Inc.). A p value < 0.05 was considered to indicate significance.

LC-HRMS and LC-MS/MS Analysis of Evogliptin and Its Metabolites
To identify evogliptin and its metabolites, an Exactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled to an Accela ultra-performance liquid chromatography system was used. The separation was performed on a Unison-C8 column (3.0 μm, 2.0 mm i.d. × 75 mm; Imtakt Corporation, Kyoto, Japan) using a gradient elution of 5% acetonitrile in 0.1% formic acid (mobile phase A) and 95% acetonitrile in 0.1% formic acid (mobile phase B) at a flow rate of 0.3 mL/min: 14% B for 8.5 min, 14% to 90% B for 3.0 min, 90% B for 3.0 min, 90% to 14% B for 0.1 min, and 14% B for 5.5 min. The column and autosampler temperatures were 40 °C and 6 °C, respectively. The electrospray ionization (ESI) in positive and negative mode was used with the following electrospray source settings: spray voltage, 4.0 kV in positive mode and −3.0 kV in negative mode; vaporizer temperature, 350 °C; capillary temperature, 330 °C; sheath gas pressure, 35 arbitrary units; and auxiliary gas pressure, 15 arbitrary units. Higher-energy collision dissociation (HCD) with a collision energy of 10 to 40 eV was employed to investigate the fragmentation pattern of evogliptin and its metabolites. The mass measurement accuracy for evogliptin and its metabolites did not exceed 5 ppm, representing a good correlation between the theoretical mass based on the molecular elemental composition and the experimental mass obtained from the full-scan HRMS analysis. The proposed structures for the product ions of evogliptin and its metabolites were determined using the Mass Frontier software (ver. 6.0; HighChem Ltd., Bratislava, Slovakia).

Data Analysis
Results represent the average of three independent experiments using human liver microsomes and human cDNA-expressed CYP and UGT enzymes. ). In the above-mentioned equations, V was the velocity of the reaction at substrate concentration [S], Vmax was the maximum velocity, Km was the substrate concentration at which the V was half of Vmax, and n is the Hill constant. The intrinsic clearance (Clint) was calculated as Vmax/Km.