In Vitro Permeability Studies.

The Caco-2 cell line (TC7-clone) was a gift of Prof. Dr. A. Zweibaum (INSERM, U-178 Villejuif, France). Cell suspensions in Dulbecco’s modified Eagle’s medium with Glutamax, 10% FBS, 1% non essential amino acids, and 1% Penicilin/Streptomycin/FBS/DMSO (v/v/v = 7:2:1) were cryopreserved and stored in liquid nitrogen in batches of approximately 2 × 10⁶ cells. Caco-2 cells used during this study were from total passage numbers 20–34. Caco-2 cell monolayers (TC7-clone) were prepared by plating 10⁵ cells/cm² in transwell plates and incubating in a CO₂-containing, water-equilibrated atmosphere at 37°C for 7 to 8 days (21 days for the paracellular permeation experiments). Tightness of monolayers was confirmed by transepithelial electrical resistance measurements in each well before and after the experiment. For investigation of its paracellular permeation, [¹⁴C]imeglimin was added to the apical compartment at various concentrations (3–300 μM) with and without EGTA (2 mM). In addition, [³H]cimetidine, a drug known to be subject to paracellular permeation, was included as a positive control in these experiments (Zhou et al., 1999). Contribution of paracellular permeation was calculated according to the following equation:where Papp is the apparent permeability and F^paracell(%) is the contribution of the paracellular permeation to the overall permeation under the in vitro conditions of the model.

A study was also performed to investigate whether imeglimin is a substrate of P-glycoprotein (P-gp). P-gp has been proven to be involved in various DDIs in humans (Ambudkar et al., 1999). To evaluate whether imeglimin is a substrate of P-gp in vitro, [¹⁴C]imeglimin was added either to basal or to apical compartments at various concentrations (1.0–1000 μM), and its time-dependent appearance in the other compartment was monitored by measuring aliquots with liquid scintillation counting (LSC). Integrity of the cell model and expression of P-gp was verified by transport experiments with digoxin, which was used as P-gp reference substrate. Testosterone was also used as a high-permeability reference substance. As imeglimin is a small cationic drug, transport through organic cation transporters (OCTs) was suspected. [¹⁴C]Imeglimin (10 µM) was incubated with or without the OCT inhibitor decynium (2 µM).

To determine whether imeglimin is a substrate of breast cancer resistance protein (BCRP), the effect of the BCRP-specific inhibitor (30 µM novobiocin) was investigated on the bidirectional permeability of imeglimin at 3 mM.

In Vitro Metabolism in Human and Rat Subcellular Fractions.

The in vitro metabolism of imeglimin at 1, 10, and 100 µM was investigated in tissue homogenates and subcellular fractions of organs and tissues known to contain cytochrome P450 and other enzyme systems capable of metabolizing xenobiotics—namely, lung, liver, kidney, and intestine. For this purpose, incubations with both S9 mix and microsomes from liver, lung, intestine, and kidney derived from human and Wistar rat were carried out. The basic incubation mixtures contained [¹⁴C]imeglimin (1, 10, and 100 μM) and 2 mM NADP, 3.3 mM glucose-6-phosphate, 0.4 U/ml glucose-6-phosphatedehydrogenase, 3.3 mM magnesium chloride, and 50 mM potassium phosphate buffer (pH 7.4), as well as either S9 (1 mg protein/ml) or microsomes (0.5 mg protein/ml). After a preincubation period (7 minutes at 37 ± 2°C), the reactions were initiated by the addition of the appropriate application solution. The final individual incubation volume was 1 ml (200 μl per time point plus additional 200 μl). Incubations were performed for 0, 15, 30, and 60 minutes at 37 ± 2°C. The reactions were then terminated by removal of a 200-μl aliquot and its addition to 0.1 ml ice-cold acetonitrile. The resulting protein precipitates in the stopped aliquots were removed in a centrifuge step at 3000 rpm. All incubations containing experimental components (including negative controls) were carried out in triplicate. The supernatants obtained after protein precipitation and centrifugation were stored frozen (at −20°C) prior to further analysis.

Binding to Plasma Protein and Blood Cell Partitioning.

The extent of in vitro protein binding and plasma/blood cell partitioning of [¹⁴C]imeglimin was determined in rat, dog, and human.

Plasma from all species was spiked with [¹⁴C]imeglimin to achieve nominal concentrations of 1, 10, and 100 μM. Triple portions were subjected to equilibrium dialysis at ∼37°C against phosphate-buffered saline (pH 7.4; Thermo Fisher Scientific, Japan) for 6 hours. After dialysis, duplicate aliquots from each cell half were analyzed by LSC to determine the concentration of radioactivity in each compartment and the percentage of protein binding. Quadruple blood samples from all species were spiked with [¹⁴C]imeglimin at two concentrations (1 and 100 μM) and incubated at ∼37°C on a rotary mixer for 4 hours. Aliquots were taken for determination of total radioactivity or transferred to hematocrit tubes for determination of the packed cell volume; the remaining blood was centrifuged at 2000g for 15 minutes, and duplicate aliquots of plasma were analyzed by LSC.

Determination of Substrate Potential with Hepatic or Renal Transporters.

Several studies were performed to characterize the in vitro affinity of imeglimin to hepatic and renal transporters.

The transport of imeglimin through OCT1 was assessed by using human embryonic kidney (HEK) 293 cells expressing solute carrier transporters. Uptake was investigated to evaluate kinetic parameters including maximal transport velocity (V_max), apparent affinity (K_m), and intrinsic uptake clearance. Imeglimin was added to the cells at 10–3000 µM and transport was stopped after 2 minutes. Metformin was used as control substrate at 10 µM.

To assess whether imeglimin is an OCT2 substrate, transport assays were performed using HEK cells stably expressing human OCT2 (hOCT2) and vector-transfected control cells. To characterize activity in hOCT2-expressing HEK cells, N-methyl-4-phenylpyridinium (MPP+) was used as a control substrate at 1 µM. Uptake by hOCT2 was investigated to evaluate kinetic parameters: V_max, K_m, and intrinsic uptake clearance. Imeglimin was added to the cells at 10–1500 µM. The cells were incubated with ¹⁴C- or ³H-labeled compounds, and accumulation within cells was quantified by LSC.

Substrate assays of imeglimin with human MATE1, MATE2-K, organic anion transporter (OAT) 1, and OAT3 were then performed. The in vitro substrate properties of imeglimin with human MATE1, MATE2-K, OAT1, and OAT3 transporters were tested at two concentrations of imeglimin (0.1 and 1 mM) at two incubation time points (2 and 20 minutes). A 1 mM maximum tested dose was selected to cover a concentration range of 50-fold versus the unbound maximum plasma concentration (7.5 µM); considering a therapeutic dose of 1000 mg, this concentration must be at least 500 µM (50 × 7.5 = 375 µM = ∼500 µM) (MHLW, 2018, (CDER, 2020b)). Metformin (10 µM), p-aminohippuric acid (5 µM), and estrone-3-sulfate (E3S at 1 µM) were used as substrates for MATE1 and MATE2-K, OAT1, and OAT3 experiments, respectively. Pyrimethamine (1 µM), benzbromarone (300 µM), and probenecid (500 µM) were used as reference inhibitors for MATE1 and MATE2-K, OAT1, and OAT3 experiments, respectively.

Cytochrome P450 Inhibition Study.

The ability of imeglimin to inhibit the major cytochrome P450 enzymes (namely, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) in vitro was evaluated with the aim of ascertaining the potential of this compound to inhibit the metabolism of concomitantly administered drugs. Inhibitory effects of imeglimin on cytochrome P450 activities were examined in human liver microsomes in the presence of NADPH.

The marker activities were as follows: CYP1A2, phenacetin O-deethylation; CYP2B6, bupropion hydroxylation; CYP2C8, paclitaxel 6α-hydroxylation; CYP2C9, diclofenac 4′-hydroxylation; CYP2C19, (S)-mephenytoin 4′-hydroxylation; CYP2D6, bufuralol 1′-hydroxylation; and CYP3A4, midazolam 1′-hydroxylation and testosterone 6β-hydroxylation. Positive control substances were as follows: CYP1A2, furafylline and α-naphthoflavone; CYP2B6, ticlopidine hydrochloride; CYP2C8, montelukast sodium and gemfibrozil 1-O-β-glucuronide; CYP2C9, sulfaphenazole and tienilic acid; CYP2C19, tranylcypromine hydrochloride and S-fluoxetine hydrochloride; CYP2D6, quinidine and paroxetine hydrochloride; and CYP3A4, ketoconazole and verapamil hydrochloride.

The tested dose must cover a concentration of 50-fold versus the unbound maximum plasma concentration (7.5 µM); considering a therapeutic dose of 1000 mg, this concentration must be 500 µM (50 × 7.5 = 375 µM = ∼500 µM) (MHLW, 2018, (CDER, 2020b)). For CYP3A4, the tested dose must cover the maximum expected concentration in the intestinal lumen (0.1-fold the maximum dose on one occasion/250 ml). The maximum concentration must be 3000 µM (0.1 × 1500 mg/250 ml = 0.1 × 31.3 mM = 3.13 mM = ∼3000 µM) (MHLW, 2018, (CDER, 2020b)). To estimate the inhibition constant (K_i) from IC₅₀, a further 2-fold greater maximum concentration (1000 and 6000 µM) should be set, as the substrate concentration is close to its K_m. The concentrations tested ranged from 0.1 to 1000 μM for all cytochrome P450 enzymes, except for CYP3A4, for which imeglimin concentrations tested were higher (up to 6000 µM). After the incubation period, marker substrate was added at a concentration approximately equal to K_m (solubility permitting), and the residual enzyme activity was measured. In addition, time-dependent inhibitory effects of imeglimin on cytochrome P450 activities with 30-minute preincubation were examined. Known metabolism-dependent inhibitors of the cytochrome P450 enzymes investigated were included as positive controls.

Cytochrome P450 Induction Studies.

Imeglimin was evaluated for its induction potential with several cytochrome P450 isoforms (CYP1A2, CYP2B6, and CYP3A4) by the mRNA expression levels of these cytochrome P450 isoforms in cryopreserved human hepatocytes from three individual donors after once-daily treatment with imeglimin at 0 (solvent control), 20, 60, and 120 µM for 48 hours. Induction potential was evaluated with the fold change in mRNA expression from solvent control and in comparison with positive control inducers. Positive control inducers 50 µM omeprazole, 2000 µM phenobarbital, and 25 µM rifampicin were used for CYP1A2, CYP2B6, and CYP3A4, respectively.

Transporter Inhibition Study.

The in vitro inhibition potential of imeglimin with the human MATE1, MATE2-K, OAT1, OAT3, organic anion–transporting polypeptide (OATP) 1B1, and OATP1B3 transporters was tested at 0.1 and 1 mM concentrations of imeglimin. The tested concentration must cover the estimated maximum concentration at the inlet to the liver for hepatic transporters (I_in,max), which is calculated as follows: I_in,max = [C_max + (Fa × Fg × ka × Dose)/Qh/R_B] × fup, where Fa is the fraction absorbed, Fg is the intestinal bioavailability, ka is the absorption rate constant, Qh is the liver blood flow, R_B is the blood-to-plasma concentration ratio and fup is is the unbound fraction in plasma. Considering the maximum therapeutic dose of 1500 mg, the concentrations should encompass 15 µM [(10 + (0.3 × 0.1 × 7826)/97/0.48) × 0.936 ∼15 µM]. The tested concentrations must cover 10 or 50 times the maximum unbound plasma concentration for OAT1, OAT3, MATE1, and MATE-2K, respectively. Considering a therapeutic dose of 1500 mg, the concentrations should encompass 100 and 500 µM (MHLW, 2018, (CDER, 2020b)). Uptake experiments were performed using Madin-Darby canine kidney cells II or HEK293 cells stably expressing the respective uptake transporters. Cells were cultured at 37 ± 1°C in an atmosphere of 95:5 air/CO₂ and were plated into standard 96-well tissue culture plates. Before the experiment, the medium was removed, and the cells were washed twice with 100 μl of assay buffer. In OAT3 experiments, cells were washed with 100 μl of HK buffer containing 5 mM glutaric acid (pH 7.4) and then were incubated at 37°C with HK buffer containing 5 mM glutaric acid (pH 7.4) for 20 minutes. Uptake experiments were carried out at 37 ± 1°C in 50 μl of assay buffer containing the probe substrate and imeglimin for 15 minutes (MATE1/MATE2-K), 2 minutes (OAT1), and 1 minute (OAT3). In the case of OATP1B1 and OATP1B3, cells were cultured at 37°C in a CO₂ incubator and plated into standard 24-well tissue culture plates. Before the experiment, cells were washed with 500 μl of 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid-Krebs-Henseleit buffer and then preincubated at 37°C for 30 minutes with preincubation medium containing imeglimin. Uptake experiments were carried out at 37°C in 250 μl of assay buffer containing the probe substrate and imeglimin or solvent for 2 minutes.

A study was performed to evaluate the inhibitory effect of imeglimin after 2 hours of incubation at 1, 2, and 3 mM on P-gp in P-gp–expressing Lilly Laboratories cell-porcine kidney 1 (LLC-PK1) cells. An imeglimin concentration of 3 mM was used to cover the maximum expected concentration in the intestinal lumen (0.1-fold the maximum dose on one occasion/250 ml) (MHLW, 2018, (CDER, 2020b)). Remaining activity was calculated from the following equations:with NFR_TC and NFR_VC corresponding to net flux ratio in the presence of the test substance and in the vehicle control, respectively.Vesicular transport assays were performed with inside-out membrane vesicles prepared from cells overexpressing human ATP-binding cassette transporters. Imeglimin was incubated with E3S (1 µM) as probe BCRP substrate for 1 minute. Ko134 (1 µM) was used as reference inhibitor.

The inhibitory potency of imeglimin toward hOCT1-expressing HEK cells was determined using [³H]MPP+ as a substrate. For IC₅₀ studies, MPP+ was used at a concentration of 0.1 µM because at this nonsaturating substrate concentration, the ratio of MPP+ concentration to the K_m of MPP+ (determined K_m = 19.9 µM) was less than 1%, and K_i is virtually identical with IC₅₀. Cells were incubated with 0.1 µM [³H]MPP+ for 2 minutes at 37°C in the presence of varying concentrations of imeglimin (10–1000 µM), and the accumulation in cells was quantified by LSC.

The inhibitory potency of imeglimin toward hOCT2-expressing HEK cells was determined using [³H]MPP+ as a substrate. Cells were incubated with 0.1 µM [³H]MPP+ for 2 minutes at 37°C in the presence of varying concentrations of imeglimin (10–1000 µM), and the accumulation in cells was quantified by LSC. The imeglimin tested concentrations cover 10-fold the maximum unbound plasma concentration (MHLW, 2018, (CDER, 2020b)).

Study Designs.

Dog and rat ADME studies were performed to assess the absorption, pharmacokinetics (PK), and routes and rates of excretion of [¹⁴C]imeglimin after oral and intravenous administration (5 mg/kg).

Additionally, based on a high-dose oral administration (100 mg/kg) to both rats and dogs, the identity of metabolites, the relative proportions of [¹⁴C]imeglimin, and its radiolabeled metabolites in plasma (only plasma for rats), urine, and feces were determined.

All studies were conducted in conformity with the guidelines. Male Wistar rats (HsdBrlHan:WIST and Hsd/Cpb:WU, 201–288 g) and a Lister-Hooded (Crl:Lis BR, 197–220 g) strain were obtained from Harlan (UK) and Charles River (UK), respectively. Female beagle dogs (7.7–8.7 kg) were obtained from Harlan (France). Dogs were fasted overnight before dosing and fed again 4 hours after administration, whereas rats were not fasted. All animals had free access to water.

Rats received a single intravenous dose (5 mg/kg) as a bolus injection via a lateral tail vein or a single oral dose (5 or 100 mg/kg) by gavage of [¹⁴C]imeglimin. For excretion balance at 5 mg/kg, the radioactive dose was in the range 6.29–6.69 MBq/kg. For pharmacokinetic and metabolite profiling, the radioactive doses used were between 1.06 MBq/kg (low dose) and 4.45 MBq/kg (high dose).

Dogs received one intravenous dose (5 mg/kg), administered via a cephalic vein, and one single oral dose (5 mg/kg) in a crossover design or a single higher oral dose (100 mg/kg) of [¹⁴C]imeglimin. Radioactive doses were 0.5 MBq/kg (low doses) and 1 MBq/kg (high dose).

For rats and dogs, oral doses were administered by gavage. For intravenous and oral administrations, the substance was dissolved in 0.9% NaCl solution.

The clinical study was a phase I, single-center, open-label, single-dose ADME study in healthy male subjects. Six subjects (aged between 30 and 60 years, inclusive, body mass index 18.5–29.9 kg/m²) received a single oral dose of 1000 mg [¹⁴C]imeglimin (containing 3.7 MBq ¹⁴C radioactivity) as an oral solution after a 10-hour overnight fast. This study was approved by an independent ethics committee (Stichting Beoordeling Ethiek Bio-Medisch Onderzoek, Assen, The Netherlands) and by the competent authority (Central Committee on Research Involving Human Subjects or, in Dutch, Centrale Commissie Mensgebonden Onderzoek) in accordance with Dutch regulations. The study was conducted in accordance with the principles of the Declaration of Helsinki and in compliance with the International Conference on Harmonization E6 Guideline for Good Clinical Practice (Committee for Proprietary Medicinal Products guideline CPMP/ICH/135/95), and compliant with the European Clinical Trial Directive: Directive 2001/20/EC. All subjects signed the informed consent form during the prescreening visit.

Sampling.

For rats, blood samples were obtained at the following time points postdose: predose, 0.1 and 0.25 (only for intravenous administration), 0.5, 1, 2, 4 and 6 (except at high oral dose), 8, 12 (except at high oral dose), 24 and 48 (except at high oral dose) hours.

At each sampling time, 0.5 ml of blood was withdrawn from the sublingual vein and transferred into heparinized tubes. The samples were centrifuged for 3 minutes at about 12,000g.

In total, 0.1 ml of the supernatant plasma was mixed with 10 ml Omni-Szintisol for determination of total radioactivity; 0.3 ml of the supernatant plasma from the high-dose animals was stored at −20°C for metabolism, and 0.3 ml of the supernatant plasma was stored at −20°C for imeglimin concentration determination. For metabolism profiling, pooled samples were prepared from four animals at the following time points: predose, 0.5, 1, 2, 8, and 24 hours after dosing. Further details for sampling are given in the Supplemental Data.

For dogs, blood samples were obtained at the following time points postdose: predose, 0.1 (only for intravenous administration), 0.25, 0.5 (except at high oral dose), 1, 2, 4 (except at high oral dose), 6, 8 (except at high oral dose), 24 and 48 (except at high oral dose) hours. Blood samples (5 to 6 ml) were withdrawn from the jugular vein into heparinized tubes and centrifuged (2500g for 10 minutes at ∼+5°C). Plasma specimens were aliquoted as follows: 0.5 ml for total radioactivity determination, 1 ml for imeglimin determination (only for low doses), 1 ml for metabolic profiling, and 1.5–2 ml for metabolite identification (only for high dose). Samples were stored at −20°C and at −80°C for metabolic profiling and metabolite identification samples. For metabolite profiling, pooled samples of plasma were prepared from three animals at the following time points: 0.25, 1, 2, and 6 hours after dosing. Urine samples were pooled by collection period: 0–8, 8–24, 24–48, and 24–48 hours. Feces samples were pooled by collection period: 0–8, 8–24, and 24–48 hours. Further details for sampling are given in the Supplemental Data. For metabolite identification, pooled samples of plasma were prepared from three animals at the following time points: 0.25, 1, 2, and 6 hours after dosing; urine, the collection periods were 0–8 and 24–48 hours; for feces, collection periods of 24–48 hours were employed. Further details for sampling are given in the Supplemental Data.

For the excretion balance in dogs and rats, urine and feces were collected into preweighed containers over the following time periods after dose administration: predose, 0–8, 8–24, 24–48, 48–72, 72–96, and 96–120 hours postdose. The total weight of the collected urine and feces was recorded.

In the clinical study, the blood sampling (12 ml) for analysis of ¹⁴C radioactivity and imeglimin was scheduled at the following time points and collected in heparin-containing tubes: predose, 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, 72, 96, 120, and 144 hours postdose. For the analysis of ¹⁴C radioactivity and imeglimin in plasma, samples were centrifuged within 30 minutes at 4°C at 1500g for 10 minutes and stored at −20°C ± 5°C until analysis. For the analysis of ¹⁴C radioactivity in blood, samples were aliquoted and stored at −20°C until analysis.

For blood-to-plasma distribution, samples of 2 ml of blood for the assessment of hematocrit were taken using potassium EDTA as the anticoagulant. Blood was collected on day 1 predose and 1, 3, and 6 hours postdose.

For metabolite pattern assessment in human plasma, 7-ml blood samples were collected in sodium heparin tubes, and the samples were immediately cooled at 4°C and centrifuged within 30 minutes at 4°C at 1500g for 10 minutes. A minimum of 3 ml of plasma was rapidly transferred to a polypropylene tube and stored immediately at −20°C ± 5°C until analysis. Blood sampling for metabolites was scheduled at the following investigation times: predose, 1, 3, 6, 8, 12, 24, 48, hours postdose. Further details for sampling are given in the Supplemental Data.

For the analysis of ¹⁴C radioactivity and metabolite patterns, urine samples were collected. After drug administration, urine was collected from all subjects according to the following time intervals: 0–4, 4–8, 8–12, 12–24, 24–36, 36–48, 48–72, 72–96, 96–120, and 120–144 hours postdose. Further details for sampling are given in the Supplemental Data.

After drug administration, feces were collected from all subjects according to the following time intervals: 0–24, 24–48, 48–72, 72–96, 96–120, and 120–144 hours postdose. All feces samples were homogenized in 24-hour portions. Further details for sampling are given in the Supplemental Data.

For the analysis of ¹⁴C radioactivity, expired-air sampling was performed in duplicate while the subject was sitting. Expired-air samples were collected into 4 ml of a trapping solution that contained 2 ml hyamine hydroxide (1 N) and 2 ml ethanol and thymolphthalein as an indicator. Breath tests were scheduled at the following investigation times: predose, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 hours postdose.

Radioactivity Distribution in Rats.

Radioactivity concentrations in tissues in rats were quantified from whole-body autoradiograms using a validated image analysis system. The legs, tail, and whiskers were trimmed off, and each frozen carcass was set in a block of frozen aqueous 2% (w/v) carboxymethylcellulose. The block was mounted onto the stage of a CM3600 cryomicrotome maintained at about −20°C [Leica Microsystems (UK) Ltd.], and sagittal sections (nominal thickness 30 μm) were obtained at five levels through the carcass: exorbital lachrymal gland (males) or ovary (females), intraorbital lachrymal gland, harderian gland and adrenal gland, thyroid, brain, and spinal cord. The sections, mounted on Invisible Tape (Supapak), were freeze-dried in a Lyolab B freeze-drier (Life Science Laboratories Ltd.) and placed in contact with FUJI imaging plates (type BAS-MS; Raytek Scientific Ltd.). The ¹⁴C blood standards of appropriate activity (also sectioned at a nominal thickness of 30 μm) were placed in contact with all imaging plates. After exposure in a refrigerated, copper-lined, lead exposure box for 4 days, the imaging plates were processed using a FUJI FLA-5000 radioluminography system (Raytek Scientific Ltd.) at a scanning resolution of 100 μm. Electronic images were analyzed using a computer-based image analysis package (Seescan Densitometry software; LabLogic Ltd., Sheffield). The carbon-14 standards included with each autoradiogram were used to construct calibration lines over a range of radioactivity concentrations.

Radioactivity Analysis.

For assessment of the excretion balance in rats, radioactivity was determined in urine, feces, cage washings (aqueous and organic), and carcasses. Volumes and/or weights of biologic samples were measured as appropriate. Dried feces homogenates, cage debris, and carcass aliquots were combusted in oxygen using a Packard Sample Oxidizer. The carbon-14 combusted products were absorbed in Carbo-SorbR and mixed with PermafluorR E+ liquid scintillant prior to LSC. Radioactivity was measured by LSC using Tri-Carb 2300 (Packard, Dreieich) liquid scintillation counters with the capability to compute quench-corrected disintegrations per minute.

In dogs, the total radioactivity content of plasma and each urine and feces pool was measured by LSC using Tri-Carb 2100 (PerkinElmer, Rodgau). Further details are given in the Supplemental Data.

For clinical samples, analysis of total radioactivity in whole blood, plasma, urine, feces, and expired air (including quick counts in urine and feces) was performed using LSC; a Packard Tri-Carb 3100 TR liquid scintillation analyzer (Downers Grove, IL) was used. Further details are given in the Supplemental Data.

Imeglimin Analysis.

The concentrations of imeglimin were investigated in plasma and urine from rats (only plasma) and dogs after single low-dose intravenous and oral administration of 5 mg/kg [¹⁴C]imeglimin and from humans after single oral 1000-mg dose by liquid chromatography with tandem mass spectrometric detection method (LC-MS/MS).

Thawed plasma samples (25 µl for animals or 50 µl for human aliquot) were transferred to a 96-well plate. Internal standard (D₆-imeglimin) solution was added, and the plate was vortex-mixed prior to transfer to a Cerex multichannel solid phase extraction system. The carboxylic acid solid phase extraction plate (50 mg) was conditioned with methanol: 20 mM sodium bicarbonate solution (90:10 v/v, 0.5 ml) followed by addition of 20 mM sodium bicarbonate solution (0.5 ml). Approximately 550 µl or 1 ml of the diluted sample was transferred to the preconditioned cartridge. The sample was then passed through the cartridge, which was subsequently washed with methanol: 20 mM sodium bicarbonate solution (90:10 v/v) (0.5 ml), followed by addition of 10 mM ammonium acetate (0.5 ml). The sample was then eluted into a fresh 96-well polypropylene collection plate with water/acetonitrile/trifluoracetic acid (10:90:0.5 v/v/v) (0.5 ml) prior to being submitted for LC-MS/MS analysis. Concentrations of imeglimin in calibration standards, quality control samples, and study samples were determined using least-squares linear regression with the reciprocal of the concentration (1/x) as weighting.

Urine samples (20-µl aliquot) were transferred into 20-ml polypropylene scintillation vials, and 100 µl of internal standard solution (20 µg/ml in water) was added. Mobile phase (8 ml) [10 mM ammonium acetate (native pH)/acetonitrile (20:80) v/v] was then added to each tube; the samples were capped and vortex-mixed, and 10 µl of each sample was aliquoted into a 96-well plate. Mobile phase (490 µl) was added to all wells, and the samples were capped and vortex-mixed again prior to submission for LC-MS/MS analysis. Concentrations of imeglimin in calibration standards, quality control samples, and study samples were determined using least-squares linear regression with the reciprocal of the concentration (1/x) as weighting. The blank sample containing internal standard was not used in the regression equation. All data are expressed as hydrochloride salt.

Metabolite Profiling.

The metabolite pattern in rat and dog in plasma was investigated after single high-dose oral administration of 100 mg/kg [¹⁴C]imeglimin. Metabolite profiling and identification was performed using liquid chromatography with radiodetection and LC-MS/MS techniques.

To identify the structures of the major metabolites in plasma and to support an exact assignment of eluting peaks to metabolite structures, a method using high-performance liquid chromatography, radiometry, and mass spectrometry was used. Structure identification was supported by comparison of retention times and fragmentation spectra of the unknown metabolites with those of the respective synthetic reference compounds. In humans, structure elucidation of metabolites was performed by time-of-flight mass spectrometry. Details are given in the Supplemental Data.

PK Analysis.

PK analysis of total radioactivity and imeglimin in plasma and blood was performed using the noncompartmental pharmacokinetic analysis using validated software (Kinetica version 4.1.1).

In rat and dog studies, the following PK parameters were reported: maximum plasma concentration (C_max), time to reach maximal concentration (t_max), and area under the plasma concentration-time curve (AUC) from time zero to last measurable concentration (AUC_0–last), AUC from time zero to infinity (AUC_0–∞), terminal elimination half-life (t_1/2), clearance of drug from plasma (CL), and volume of distribution (V_ss). The bioavailability and fraction absorbed were determined according to the following equations:For clinical study, the following PK parameters were reported for blood and plasma total radioactivity and plasma imeglimin: C_max, t_max, AUC_0–last, AUC_0–∞, t_1/2, apparent clearance of drug from plasma, and apparent volume of distribution (V_ss/F).

For each collection interval time, the cumulative amount excreted in urine was calculated as the product of urine concentration and urine volume. CL_R was calculated over the period from 0–48 hours according to the following formula:For metabolites, the concentrations of total radioactivity of parent drug and metabolites were measured, and individual AUC_0–last values were calculated; subsequently, the %AUC_TR (AUC of total radioactivity) was calculated using the following formula: