http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, Early Online: 1–10 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.999141
RESEARCH ARTICLE
Biotransformation and mass balance of the SGLT2 inhibitor empagliflozin in healthy volunteers Lin-Zhi Chen1, Arvid Jungnik2, Yanping Mao1, Elsy Philip1, Dale Sharp1, Anna Unseld2, Leo Seman1, Hans-Ju¨rgen Woerle3, and Sreeraj Macha1 1
Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA, 2Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany, and Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim, Germany
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Abstract
Keywords
1. The absorption, biotransformation and excretion of empagliflozin, an SGLT2 inhibitor, were evaluated in eight healthy subjects following a single 50 mg oral dose of empagliflozin containing 100 mCi [14C]-empagliflozin. 2. Radioactivity was rapidly absorbed, with plasma levels peaking 1 h post-dose. Total exposure was lower in blood versus plasma, consistent with moderate (28.6–36.8%) red blood cell partitioning. Protein binding was 80.3–86.2%. 3. Most of the radioactive dose was recovered in urine (54.4%) and faeces (41.1%). Unchanged empagliflozin was the most abundant drug-related component in plasma, representing 75.5–77.4% of plasma radioactivity and 79.6% plasma radioactivity AUC0–12 h. Unchanged empagliflozin was the most abundant drug-related component in urine and faeces, representing 43.5% (23.7% of dose) and 82.9% (34.1% of dose) of radioactivity in urine and faeces, respectively. Six metabolites were identified in plasma: three glucuronide conjugates representing 4.7–7.1% of AUC0–12 h and three less abundant metabolites (50.2–1.9% AUC0–12 h). The most abundant metabolites in urine were two glucuronide conjugates (7.8–13.2% of dose) and in faeces was a tetrahydrofuran ring-opened carboxylic acid metabolite (1.9% of dose). 4. To conclude, empagliflozin was rapidly absorbed and excreted primarily unchanged in urine and faeces. Unchanged parent was the major drug-related component in plasma. Metabolism was primarily via glucuronide conjugation.
Absorption, excretion, metabolic pathway, metabolite, pharmacokinetics, radiolabeled
Introduction Type 2 diabetes mellitus (T2DM) is a chronic disease characterised by hyperglycaemia and progressive beta-cell failure (DeFronzo, 2009). The prevalence and incidence of T2DM are increasing worldwide (International Diabetes Federation, 2013). In the US and Europe, T2DM is a leading cause of cardiovascular disorders, blindness, end-stage renal disease and amputations (Inzucchi et al., 2012). The ultimate aim of managing patients with T2DM is to control glycaemia and thus prevent or delay the development of complications (Inzucchi et al., 2012). However, despite the availability of many anti-diabetes agents, many patients fail to achieve recommended glycaemic targets (Esposito et al., 2012). The sodium glucose cotransporter 2 (SGLT2), located in the proximal tubule, is the transporter primarily responsible
Address for correspondence: Lin-Zhi Chen, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA. Tel: + 1 (203) 7787870. E-mail: [email protected]
History Received 21 October 2014 Revised 10 December 2014 Accepted 12 December 2014 Published online 30 December 2014
for reabsorption of the glucose filtered by the kidney (DeFronzo et al., 2012). In healthy individuals, virtually all of the 180 g/d of glucose filtered by the kidney is reabsorbed into the bloodstream (Gerich, 2010). Inhibition of SGLT2 reduces renal glucose reabsorption and increases urinary glucose excretion (UGE), thereby reducing hyperglycaemia in patients with T2DM (DeFronzo et al., 2012). Empagliflozin is a potent and selective SGLT2 inhibitor (Grempler et al., 2012) developed as a treatment for T2DM. The pharmacokinetics of empagliflozin are similar in healthy subjects and patients with T2DM (Heise et al., 2013a,b; Seman et al., 2013). After oral administration of an empagliflozin tablet, empagliflozin is rapidly absorbed, with peak plasma concentrations occurring a median 1.5 h postdose (Heise et al., 2013a). Thereafter, plasma concentrations decline in a biphasic manner with a rapid distribution phase and a relatively slow terminal phase. The single-dose and steady-state pharmacokinetics of empagliflozin are similar, suggesting linear pharmacokinetics with respect to time; systemic exposure to empagliflozin increases in a doseproportional manner (Heise et al., 2013a,b).
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Empagliflozin provides a sustained increase in UGE in patients with T2DM (Heise et al., 2013a). In phase III trials in patients with T2DM, empagliflozin, given as monotherapy or as add-on to other anti-diabetes therapies, was consistently shown to improve glycaemic control, to reduce body weight and blood pressure, and to be well-tolerated, with a low risk of hypoglycaemia (Barnett et al., 2014; Haering et al., 2013, 2014; Kovacs et al., 2014; Roden et al., 2013). The current study was undertaken to determine the metabolism, pharmacokinetics and excretion of a single oral dose of [14C]-empagliflozin in healthy subjects.
Materials and methods
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Subjects Eight healthy male non-smokers were enrolled. Inclusion criteria included age 18–55 years and body mass index (BMI) of 18.5–29.9 kg/m2. Exclusion criteria included medical or laboratory findings of clinical relevance, Gilbert’s syndrome, irregular defecation pattern (51 bowel movement/day); history of gastrointestinal surgery (except appendectomy), orthostatic hypotension, fainting, blackouts, hypersensitivity, habitual tobacco/nicotine use (within prior 6 months) or alcohol abuse; blood donation within 4 weeks of, or during, the trial; participation in more than one study with a radiolabelled investigational drug in the last year; or administration of a radiolabelled drug within the last 6 months. The use of prescription or over-the-counter drugs and herbal preparations was restricted within 14 d of study drug administration, as was the use of drugs with a half-life >24 h within 1 month. All subjects provided written informed consent prior to participation. Study design This was a phase I, open-label, single-centre, single-dose study. Subjects received 50 mg empagliflozin containing [14C]-radiolabelled empagliflozin in solution following an overnight fast of 10 h. The empagliflozin solution was administered via syringe directly into the mouth, after which the subject drank 240-mL water. The dose of empagliflozin administered to each subject was calculated based on the difference in the weight of the syringe before and after dose administration. Fasting continued for 4 h after dosing. Standard meals were served 4.5, 8 and 10.5 h after study drug administration. There were no special requirements regarding food intake on other study days. Subjects remained in the study centre for 7 and 15 d after drug administration; they were discharged when 90% of the administered radioactivity had been recovered in the urine and faeces or 1% of the administered radioactivity was recovered in total in the urine and faeces over two consecutive 24-h periods. The study protocol was approved by the local Institutional Review Board and complied with the Declaration of Helsinki in accordance with the International Conference on Harmonisation Harmonised Tripartite Guideline for Good Clinical Practice. Test compound and chemicals Empagliflozin powder for oral solution and [14C]-empagliflozin powder were manufactured by Boehringer Ingelheim
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Pharmaceuticals, Inc. (Ridgefield, CT). [14C]-empagliflozin material had a radiopurity of >97%, as determined by highperformance liquid chromatography (HPLC) coupled with flow scintillation detection. A mixture of empagliflozin and [14C]-empagliflozin was reconstituted with polyethylene glycol 400/water (60/40, w/w) at a concentration of 5 mg/g to provide a radioactive dose of 100 mCi per subject (49.1 mg empagliflozin plus 0.9 mg [14C]-empagliflozin, adjusted according to the specific activity). The specific activity of [14C]-empagliflozin was 2.13 mCi/mg. Three empagliflozin glucuronide standards (6-O-glucuronide, 2-O-glucuronide and 3-O-glucuronide conjugates) were synthesised by Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT). The liquid scintillation cocktails Ultima Gold, Ultima FLO-MÕ and the absorbent Carbo-Sorb were obtained from PerkinElmer (Meriden, CT). All other chemicals and solvents were of HPLC or analytical grades and obtained from reliable commercial sources. Sample collection Blood samples were collected at the following intervals relative to study drug administration for pharmacokinetic analyses of empagliflozin in plasma, and radioactivity in blood and plasma: pre-dose, 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 h. Metabolite profiling and haematocrit (for evaluation of RBC partitioning: Cblood cells/Cplasma ratio of radioactivity) were conducted on samples collected 1 h prior to dose (for haematocrit only) and 2, 6 and 12 h post-dose. Plasma protein binding was determined using samples collected before (1 h) and 1.5 and 3 h after study drug administration. Urine samples were collected to determine the concentrations of empagliflozin and radioactivity in urine, and to investigate the metabolic profile of empagliflozin. A blank urine sample was collected 2 h prior to study drug administration. Urine was collected during the following intervals after study drug administration: 0–4, 4–8, 8–12, 12–24, 24–28, 28–32, 32–36 h and then in 24-h intervals until the end of the study. A faecal sample was collected within 24 h prior to study drug administration, if possible. All faeces were then collected over 24-h time intervals up to 168 h post-dose for the analysis of radioactivity and metabolic profiling. The weight of the samples was recorded. Safety assessments Safety was assessed through monitoring of vital signs (blood pressure and pulse rate), 12-lead electrocardiogram (ECG), clinical laboratory tests and adverse events (AEs) and via a global tolerability assessment (good, satisfactory, not satisfactory, or bad) undertaken by the investigator at discharge. Bioanalytical methods Empagliflozin in plasma and urine Validated HPLC methods with tandem mass spectrometric (MS/MS) detection were used to determine empagliflozin concentrations in plasma and urine using K3EDTA as an
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anti-coagulant. Empagliflozin and the internal standard, [13C6]-empagliflozin, were extracted from plasma and urine using solid-supported liquid extraction. After evaporation under nitrogen, the residue was reconstituted and analysed using liquid chromatography (LC) with MS/MS. The LC/ MS/MS method quantitated only the unlabelled portion of the drug. As unlabelled drug accounted for >99% of the total dose, the concentrations of unlabelled drug were treated as the total drug concentrations, with no correction for the labelled drug. The lower limit of quantification for empagliflozin in plasma and urine was 1.11 nM and 4.44 nM, respectively, with linearity to 1110 nM and 4440 nM, respectively.
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Radioactivity measurement Whole blood, plasma, urine and faeces were analysed for radioactivity by liquid scintillation counting (LSC). Each sample was homogenised prior to radioanalysis. Faecal samples were diluted in water prior to homogenisation. Plasma and urine samples were analysed directly; blood and faecal samples were combusted (Model 307 Sample Oxidizer, Perkin Elmer, Waltham, MA), and the resulting 14CO2 trapped in a mixture of Perma Fluor and Carbo-Sorb prior to LSC. All samples were analysed using Model 2900T LSC (Perkin Elmer, Waltham, MA) using Ultima Gold XR scintillation cocktail for 5 min or 100 000 counts. All samples were analysed in duplicate. Pharmacokinetic analysis Non-compartmental pharmacokinetic parameters for empagliflozin and total radioactivity were determined using WinNonLinTM software (version 5.2, Pharsight Corporation, Sunnyvale, CA). Maximum plasma concentrations (Cmax) and time to Cmax (tmax) values were determined directly from the plasma concentration–time curves. The terminal half-lives (t1/2) were calculated as the quotient of ln(2) and the apparent terminal rate constant (lz), where lz was estimated from a regression of ln(C) versus time over the terminal loglinear disposition portion of the concentration-time profiles. AUC0–1 was estimated as the sum of the area under the curve (AUC) over the time interval 0 to the time of the last quantifiable data point with the extrapolated area given by the quotient of the last measured concentration and lz. The fraction of dose excreted in the urine (feurine) was calculated as the quotient of the sum of drug/radioactivity excreted in the urine over all observed intervals and the dose administered, and the fraction of the dose of total radioactivity excreted in the faeces (fefaeces) was calculated in a similar way. Plasma protein binding of radioactivity was determined using equilibrium dialysis for 6 h at 37 C. The ratio of concentration of total radioactivity in blood cells and plasma (Cblood cells/Cplasma) was calculated according to the following formula, where Cblood cells is the concentration in blood cells, Cplasma is the concentration in plasma, and HC is the haematocrit (decimal; determined using standard laboratory procedures): Cblood cells =Cplasma ¼
ðCblood cells Cplasma Þ ð1 HCÞ : HC Cplasma
Biotransformation and mass balance of empagliflozin
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Metabolite sample extraction The 2-h and 6-h plasma samples were profiled individually. Due to low plasma radioactivity, the 12-h samples from all subjects were combined. An equal volume of water was added to every sample. The diluted samples were subjected to solidphase extraction (SPE) with preconditioned Oasis MCX SPE cartridges (35 cc/6 g for the 2-h and 12-h samples; 20 cc/1 g for 6-h samples; Waters, Milford, MA). After loading, the SPE cartridges were washed sequentially with 0.5% acetic acid, water and 5% methanol in water and the sample was eluted with methanol. The eluate was evaporated to 0.2 mL under a nitrogen stream at 35 C (Zymark Turbovap LV evaporator, GenTech Scientific Inc., Hopinton, MA). The final samples were reconstituted in methanol and water (50:50) and transferred to autosampler vials. The overall recovery of radioactivity from plasma was 85.3–99.8%. Urine collected until 48-h post-dose was pooled for each subject; this sample represented 94.4–98.4% of the total radioactivity ultimately recovered in urine. A 100 mL aliquot of pooled urine was extracted (Oasis MCX SPE cartridge 35 cc/6 g; Waters, Milford, MA), washed, eluted, dried (to 0.1 mL) and reconstituted as for plasma samples. The overall recovery of radioactivity from urine was 87.8–100%. Faeces collected until 144-h post-dose was pooled for each subject. This represented 94.2–99.8% of total radioactivity ultimately recovered in faeces. A 10 mL aliquot of the pooled faecal homogenate was centrifuged at 10 000 rpm for 15 min at 10 C. The supernatant was separated and the pellet extracted with methanol (2) and 10% acetic acid in methanol. The extracts were combined and then evaporated to dryness under a nitrogen stream at 35 C. The residue was sonicated to dissolve in 20 mL water and then combined with the supernatant. The combined sample was extracted (Oasis MCX SPE cartridges 35 cc/6 g; Waters, Milford, MA), washed, eluted, dried (to 0.1–0.2 mL) and reconstituted (with 1 mL methanol in water, 50:50) as for plasma and urine samples. The overall extraction recovery from faeces was 82.7–100%. Metabolite profiling and identification Metabolite profiling and identification in plasma, urine and faeces were conducted using an LC/radiochromatography/ MS/MS system. Empagliflozin metabolites were identified based on HPLC retention time, radiochromatography and MS. MS, MS/MS, triple mass spectrometry (MS3) and accurate mass measurements were performed for metabolite structure elucidation. The system comprised an Agilent 1200 HPLC system (Palo Alto, CA), with a Thermo LTQ Orbitrap XL mass spectrometer (San Jose, CA) and a PerkinElmer 625 TR flow scintillation analyser (Shelton, CT) serving as detectors. Radiochromatograms generated from the flow scintillation analyser were processed using ProFSA software (PerkinElmer, Shelton, CT). The LTQ Orbitrap mass spectrometer was operated with Xcalibur 2.0 software (Thermo Scientific, San Jose, CA, USA). Thermo MetWorks 1.1 software (Thermo Scientific, San Jose, CA) with mass defect filter was used to assist metabolite identification. Authentic standards for empagliflozin and its three glucuronide conjugates were used for confirmation of identification.
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Results
Radioactivity in blood and plasma
Subjects
The shapes of the profiles for radioactivity in blood and plasma were similar (Figure 2). Radioactivity was rapidly absorbed, reaching peak plasma levels 1 h after administration, after which plasma concentrations declined in a biphasic manner with a rapid distribution phase and a slower elimination phase. Total radioactivity levels were below the limit of quantification at 36 h post-dose in the blood and at 48 h post-dose in the plasma. Radioactivity exposure was
Eight subjects aged 21–53 years with a mean BMI of 25.0 kg/m2 (range 21.0–28.9 kg/m2) and a mean body weight of 79 kg were enrolled and received study drug. One or more AE was reported in five subjects. All AEs were mild in intensity. Gastrointestinal AEs (abdominal distension, flatulence) experienced by three subjects were considered drug-related by the investigator. The investigator considered the global tolerability of empagliflozin to be good for all eight subjects. Administration of empagliflozin had no effect on vital signs or ECG and no consistent or clinically relevant effect on clinical laboratory findings.
Table 1. Pharmacokinetics of radioactivity and empagliflozin after a single dose of empagliflozin 50 mg containing 100 mCi [14C]-empagliflozin in eight healthy subjects. Radioactivity Blood (n ¼ 8)
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Mass balance The overall recovery of radioactivity in the urine and faeces was 95.6%(range: 93.0–99.4%) over 168 h after administration of study drug. Most (94.3%) of the radioactivity was recovered in the first 120 h. Figure 1 shows the cumulative amounts of radioactivity excreted in the urine and faeces as a percentage of the dose and in nM. A mean of 54.4% of the radioactive dose was excreted in the urine and 41.2% in the faeces (Figure 1; Table 1). Figure 1. Cumulative amounts of radioactivity excreted in the urine and faeces (a) as % of dose and (b) as nmol following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin to eight healthy subjects.
Plasma (n ¼ 8)
Empagliflozin Plasma (n ¼ 8)
16 100 ± 20.9 15 600 ± 19.5 AUC0–1 (nmolh/L) 11 000 ± 19.5 Cmax (nmol/L) 1470 ± 21.3 2200 ± 21.4 2260 ± 20.9 tmax (h) 1.0 (0.8–1.5) 1.0 (0.8–1.0) 0.9 (0.8–1.0) t½ (h) 8.7 ± 12.8 9.2 ± 6.9 15.9 ± 46.3 CLR,0–tz (mL/min) 63.7 ± 21.2 34.3 ± 27.9 feurine,0–tz (%) 54.4 ± 6.4 28.6 ± 13.6 fefaeces,0–tz (%) 41.2 ± 7.2 Not measured Data are mean ± %CV except tmax, which is median (range).
Biotransformation and mass balance of empagliflozin
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Figure 2. Concentration versus time profiles for radioactivity in blood and plasma and empagliflozin in plasma following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin to eight healthy subjects.
Table 2. Mean radioactivity contributions of empagliflozin and its metabolites after administration of a single dose of empagliflozin 50 mg containing 100 mCi [14C]-empagliflozin in eight healthy subjects. % of Sample radioactivity [concentration(nM) in plasma] Compound Empagliflozin M626/3 M626/1 M626/2 M482/1 M464/1 M468/1
2h 77.4 7.4 6.2 3.7 1.2 0.5 0.4
[1320] [127] [109] [62.8] [24.3] [8.5] [5.6]
6h 75.5 6.3 5.0 6.0 1.8 0.4 0.2
[638] [53.4] [42.1] [49.4] [17.1] [3.1] [1.5]
% AUC0–12 h
12 h 76.2 5.4 5.2 3.3 3.1 1.1
[283] [20.1] [19.3] [12.3] [11.5] [4.1] –
% of Sample radioactivity [% dose]
nM h/L
% Total
Faeces
7999 708.3 595.9 472.3 192.9 53.3 24.3
79.6% 7.1% 5.9% 4.7% 1.9% 0.5% 0.2%
82.9 [34.2] – – – 4.6 [1.9] 2.6 [1.1] 1.4 [0.6]
lower in blood than plasma (Figure 2; Table 1), consistent with moderate partitioning of radioactivity into RBC. Mean RBC:plasma concentration ratios of radioactivity were 28.6%, 30.2% and 36.8% at 2, 6 and 12 h post dose. Protein binding of radioactivity varied little across time points and ranged from 80.3–86.2%. Empagliflozin in plasma and urine The rates of absorption and disposition of total radioactivity and empagliflozin were similar (Figure 2; Table 1). There were no major differences in the pharmacokinetics of total radioactivity and empagliflozin (Table 1). When empagliflozin and radioactivity exposure were compared, the majority of total radioactivity in plasma appears to be unchanged empagliflozin. A total of 28.6% of the empagliflozin dose was excreted unchanged in the urine. Metabolite profiles Unchanged empagliflozin was the most abundant drug-related component in the plasma, urine and faeces (Table 2; Figures 3a–c). No major metabolites were detected in plasma. Empagliflozin accounted for 75.5–77.4% of plasma radioactivity or 79.6% of plasma radioactivity AUC0–12 h. Six minor metabolites were identified in plasma. Three glucuronide conjugates (M626/1, M626/2, M626/3) were identified using authentic standards (Figure 3a) and
Urine 43.5 24.1 14.4 3.9 5.2 1.5
[23.7] [13.2] [7.8] [2.1] [2.8] [0.9] –
Total % dose (urine + faeces) 57.9 13.2 7.8 2.1 4.7 2.0 0.6
represented 3.7–7.4% of plasma radioactivity and 4.7–7.1% of AUC0–12 h. Three less abundant metabolites were tentatively identified and represented 51.9% of AUC0–12 h: a tetrahydrofuran ring-opened carboxylic acid metabolite (M482/1), an oxidation/dehydrogenation metabolite (M464/1) and a tetrahydrofuran ring-opened dihydroxyl metabolite (M468/1) (Table 2). The metabolic pathways and associated structures are presented in Figure 4. Urinary excretion accounted for 54.4% of the radioactive dose (Table 1). Empagliflozin accounted for 43.5% of the radioactivity detected in urine (23.7% of the dose). The remaining radioactivity was accounted for primarily by five metabolites, all of which were also observed in the plasma (the three glucuronide conjugates, M482/1 and M464/1) (Table 2, Figure 3b). Faecal excretion accounted for 41.2% of the radioactive dose (Table 1). Empagliflozin accounted for 82.9% of the radioactivity detected in faeces (34.2% of the dose). The remaining faecal radioactivity was accounted for primarily by the three less abundant metabolites, which were also found in the plasma (Table 2, Figure 3c). Metabolite identification Empagliflozin Empagliflozin corresponded to the radioactive peak eluting at 65.2 min in plasma, urine and faeces, as it had the same
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Figure 3. Radiochromatograms of 2-h plasma (a), urine (b) and faeces (c) from subject M107 following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin.
retention time, MS and MS/MS patterns as the authentic standard. The molecular ions at m/z 451 (positive mode) and m/z 449 (negative mode) with an isotope ratio of 0.35 for [M + 2]/M was characteristic of the presence of one chlorine atom. MS/MS produced several abundant fragment ions in positive mode, the majority of which were also observed in negative mode, which showed the loss of the tetrahydrofuran ring, corresponding to the m/z 379 ion (Figure 5).
M464/1 A radioactive peak eluting at 62.1 min was observed in plasma, urine and faeces. Positive MS showed an elemental composition of C23H26O8Cl. MS/MS was consistent with an apparent oxidation/dehydrogenation metabolite (Figure 7). M468/1
Radioactive peaks eluting at 49.5, 54.3 and 60.9 min were found in plasma and urine and had the same retention, molecule weight, elemental composition and MS/MS spectra as respective authentic standards of the three glucuronide conjugates of empagliflozin. They had similar MS/MS results with characteristic empagliflozin fragments at m/z 395, 379, 371, 359 and 329.
A radioactive peak eluting at 56.7 min was observed in plasma and faeces, corresponding to a negative molecular ion at m/z 467, 18 Da more than empagliflozin. High resolution MS indicated that the 18 Da were from one oxygen and two hydrogen atoms. The isotope ratio of [M + 2]/[M] was 0.35, suggesting that the chlorine atom was retained. Negative MS/MS gave a predominant fragment at m/z 379 (Figure 8). MS3 for this fragment was dominated by fragments at m/z 301, 289 and 259. Mass spectral results were consistent with a tetrahydrofuran ring-opened dihydroxyl structure.
M482/1
Discussion
A radioactive peak eluting at 40.4 min was observed in plasma, urine and faeces corresponding to a negative molecular ion at m/z 481, 32 Da more than empagliflozin. The isotope ratio of [M + 2]/[M] was 0.35, suggesting that the chlorine atom was retained. High resolution MS yielded the most likely formula, C23H28O9Cl. Negative MS/MS gave fragments at m/z 379, 361, 301, 319 and 259, suggesting that the 32 Da were added to the furan ring (Figure 6). Based on the mass spectral analyses, M482/1 was tentatively identified as a tetrahydrofuran ring-opened carboxylic metabolite.
Empagliflozin was rapidly absorbed, underwent limited metabolism and was excreted primarily unchanged in the urine and faeces. The most abundant metabolites in plasma were three glucuronide conjugates (2-O, 3-O and 6-O glucuronide). Systemic exposure of each metabolite was less than 10% of total drug-related material. In addition, the PK profile including AUC0–1, Cmax, tmax and t1/2 was similar between empagliflozin and total plasma radioactivity, indicating minimal metabolite accumulation at steady state upon multiple dosing. There were no major human metabolites that
M6261/1, M626/2 and M626/3
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Figure 4. Metabolic pathway for empagliflozin following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin to healthy subjects.
required non-clinical safety assessments (U.S. Department of Health and Human Services, 2008). Similar to plasma, unchanged empagliflozin was the major drug-related component in excreta and accounted for 57.9% of the dose in urine and faeces combined. The major elimination pathway for empagliflozin was via the urine and it is possible that active secretion could account for a portion of the empagliflozin excreted unchanged in the urine. Empagliflozin is a substrate of the human organic anion transporter 3 (OAT3), which is expressed on the basolateral membrane of the renal proximal tubule and is involved in the renal excretion of many drugs (data on file). Thus, OAT3 could be responsible for the active secretion of empagliflozin into the urine. The biotransformation of empagliflozin in humans mainly involved glucuronide conjugation and, to a lesser extent, oxidation. The glucuronidation took place on the glucose moiety to produce metabolites M626/1, M626/2 and M626/3. Oxidation on the tetrahydrofuran ring followed by ring opening led to the formation of dihydroxyl metabolite M468/1 and carboxylic acid metabolite M482/1, possibly via an aldehyde intermediate (not detected). Oxidation was also responsible for the formation of M464/1. All these empagliflozin metabolites have also been seen in rats, mice or dogs (data on file). Compared to humans, metabolic
pathways involving oxidation were much more prominent in the animals and several other oxidation metabolites were found in the animals. The biotransformation of empagliflozin is different to that of the SGLT2 inhibitors canagliflozin and dapagliflozin. While unchanged canagliflozin is the most abundant drugrelated component in plasma, representing 60% of plasma radioactivity, it has two major metabolites, both glucuronide conjugates, which represent 19% and 16% of radioactivity in plasma (Mamidi et al., 2014). Canagliflozin is primarily excreted in the faeces, with 60% of the radioactive dose in faeces and 33% in urine. Unchanged canagliflozin is the primary drug-related component in faeces, representing 39% of the radioactive dose. Unchanged canagliflozin is not present in the urine, only the metabolites which represent 14% and 18% of the radioactive dose (Mamidi et al., 2014). In contrast to empagliflozin and canagliflozin, dapagliflozin is extensively metabolised. A ratio of dapagliflozin concentration to total plasma radioactivity of 0.3 suggests that metabolites account for a significant proportion of drug-related components in plasma (Obermeier et al., 2010). A major metabolite, dapagliflozin 3-O-glucuronide, is found at similar concentrations to dapagliflozin in plasma; together these account for >72% of plasma
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Figure 5. Positive MS/MS of empagliflozin [M + H]+ ion at m/z 451 (a) and negative MS/MS of empagliflozin [M H] ion at m/z 449 (b) from authentic standard.
Figure 6. Negative MS/MS of M482/1 molecular ion at m/z 481 from the urine of subject M115 following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin.
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Figure 7. Positive MS/MS of M464/1 molecular ion at m/z 465 from the faeces of subject M107 following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin.
Figure 8. Negative MS/MS of M468/1 molecular ion at m/z 467 from the faeces of subject M115 following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin.
radioactivity (Obermeier et al., 2010). The formation of dapagliflozin 3-O-glucuronide is mediated by uridine diphosphate glucuronosyltransferase (UGT) 1A9, an enzyme present in the liver and kidney, with CYP-mediated metabolism as a minor clearance pathway (Bristol-Myers Squibb/AstraZeneca, 2014). Dapagliflozin is primarily excreted in the urine, with 75% of the radioactive dose excreted in urine and 21% in faeces (Bristol-Myers Squibb/ AstraZeneca, 2014; Obermeier et al., 2010). Unchanged dapagliflozin represents 1.6% of the radioactive dose in the urine and 15% in the faeces (Bristol-Myers Squibb/ AstraZeneca, 2014; Obermeier et al., 2010). The results of this study demonstrated that the major elimination pathway for empagliflozin is via the urine. In light of this, a study was undertaken to determine the effect of renal impairment on the pharmacokinetics of a single 50 mg dose of empagliflozin. This study found that there were no clinically relevant increases in exposure in patients
with renal impairment compared to patients with normal renal function, suggesting that no dose adjustments of empagliflozin are required in patients with renal impairment (Macha et al., 2014a). Similarly, a hepatic impairment study conducted to investigate whether decreased hepatic metabolism of empagliflozin might increase exposure found that hepatic impairment had no clinically relevant effects on drug exposure, suggesting that no dose adjustment of empagliflozin is required in patients with hepatic impairment (Macha et al., 2014b).
Conclusion Following a single oral dose in healthy subjects [14C]empagliflozin was rapidly absorbed and excreted primarily unchanged in the urine and faeces. A total of 54.4% of the empagliflozin dose was excreted in urine and 41.2% in the faeces. Empagliflozin underwent limited metabolism, as
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demonstrated by unchanged parent being the predominant drug-related component in the plasma, urine and faeces. Six minor metabolites (each less than 10% of total drug-related material) were identified. Radioactivity exposure was lower in blood than plasma, consistent with moderate partitioning into RBC.
Acknowledgements The authors acknowledge the contribution of Debra Mandarino, MD, who was the principal investigator at the clinical site (Covance Laboratories, Madison, WI).
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Declaration of interest Medical writing assistance, supported financially by Boehringer Ingelheim, was provided by Clare Ryles and Wendy Morris of Fleishman-Hillard Group, Ltd, during the preparation of this article. This study was funded by Boehringer Ingelheim. All authors are employees of Boehringer Ingelheim. The authors were fully responsible for all content and editorial decisions and were involved at all stages of article development and have approved the final version.
References Barnett AH, Mithal A, Manassie J, et al. (2014). Efficacy and safety of empagliflozin added to existing antidiabetes treatment in patients with type 2 diabetes and chronic kidney disease: a randomised, doubleblind, placebo-controlled trial. Lancet Diabetes Endocrinol 2:369–84. Bristol-Myers Squibb/AstraZeneca. Forxiga (dapagliflozin) summary of product characteristics. (2014). Available from: http://www.ema.europa.eu/ema/index.jsp?curl¼pages/medicines/human/medicines/ 002322/human_med_001546.jsp&mid¼WC0b01ac058001d124 [last accessed 15 Sept 2014]. DeFronzo RA. (2009). Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes 58:773–95. DeFronzo RA, Davidson JA, del Prato S. (2012). The role of the kidneys in glucose homeostasis: a new path towards normalizing glycaemia. Diabetes Obes Metab 14:5–14. Esposito K, Chiodini P, Bellastella G, et al. (2012). Proportion of patients at HbA1c target 57% with eight classes of antidiabetic drugs in type 2 diabetes: systematic review of 218 randomized controlled trials with 78945 patients. Diabetes Obes Metab 14:228–33. Gerich JE. (2010). Role of the kidney in normal glucose homeostasis and in the hyperglycaemia of diabetes mellitus: therapeutic implications. Diabet Med 27:136–42. Grempler R, Thomas L, Eckhardt M, et al. (2012). Empagliflozin, a novel selective sodium glucose cotransporter-2 (SGLT-2) inhibitor: characterisation and comparison with other SGLT-2 inhibitors. Diabetes Obes Metab 14:83–90.
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Haering HU, Merker L, Seewaldt-Becker E, et al. (2013). Empagliflozin as add-on to metformin plus sulfonylurea in patients with type 2 diabetes: a 24-week, randomized, double-blind, placebo-controlled trial. Diabetes Care 36:3396–404. Haering HU, Merker L, Seewaldt-Becker E, et al. (2014). Empagliflozin as add-on to metformin in patients with type 2 diabetes: a 24-week, randomized, double-blind, placebo-controlled trial. Diabetes Care 37: 1650–9. Heise T, Seewaldt-Becker E, Macha S, et al. (2013a). Safety, tolerability, pharmacokinetics and pharmacodynamics following 4 weeks’ treatment with empagliflozin once daily in patients with type 2 diabetes. Diabetes Obes Metab 15:613–21. Heise T, Seman L, Macha S, et al. (2013b). Safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple rising doses of empagliflozin in patients with type 2 diabetes mellitus. Diabetes Ther 4:331–45. International Diabetes Federation. (2013). IDF diabetes atlas. Executive Summary. Available from: http://www.idf.org/sites/default/files/ EN_6E_Atlas_Exec_Sum_1.pdf [last accessed 15 Sept 2014]. Inzucchi SE, Bergenstal RM, Buse JB, et al. (2012). Management of hyperglycemia in type 2 diabetes: a patient-centered approach: position statement of the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetes Care 35:1364–79. Kovacs CS, Seshiah V, Swallow R, et al. (2014). Empagliflozin improves glycaemic and weight control as add-on therapy to pioglitazone or pioglitazone plus metformin in patients with type 2 diabetes: a 24week, randomized, placebo-controlled trial. Diabetes Obes Metab 16: 147–58. Macha S, Mattheus M, Halabi A, et al. (2014a). Pharmacokinetics, pharmacodynamics and safety of empagliflozin, a sodium glucose cotransporter 2 (SGLT2) inhibitor, in subjects with renal impairment. Diabetes Obes Metab 16:215–22. Macha S, Rose P, Mattheus M, et al. (2014b). Pharmacokinetics, safety and tolerability of empagliflozin, a sodium glucose cotransporter 2 inhibitor, in patients with hepatic impairment. Diabetes Obes Metab 16:118–23. Mamidi RN, Cuyckens F, Chen J, et al. (2014). Metabolism and excretion of canagliflozin in mice, rats, dogs, and humans. Drug Metab Dispos 42:903–16. Obermeier M, Yao M, Khanna A, et al. (2010). In vitro characterization and pharmacokinetics of dapagliflozin (BMS-512148), a potent sodium-glucose cotransporter type II inhibitor, in animals and humans. Drug Metab Dispos 38:405–14. Roden M, Weng J, Eilbracht J, et al. (2013). Empagliflozin monotherapy with sitagliptin as an active comparator in patients with type 2 diabetes: a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Diabetes Endocrinol 1:208–19. Seman L, Macha S, Nehmiz G, et al. (2013). Empagliflozin (BI 10773), a potent and selective SGLT-2 inhibitor, induces dose-dependent glucosuria in healthy subjects. Clinical Pharm in Drug Dev 2:152–61. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Food and Drug Administration. (2008). Guidance for Industry: Safety Testing of Drug Metabolites. Available from: http://www.fda.gov/ downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/ucm079266.pdf [last accessed 15 Sep 2014].
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RESEARCH ARTICLE
Biotransformation and mass balance of the SGLT2 inhibitor empagliflozin in healthy volunteers Lin-Zhi Chen1, Arvid Jungnik2, Yanping Mao1, Elsy Philip1, Dale Sharp1, Anna Unseld2, Leo Seman1, Hans-Ju¨rgen Woerle3, and Sreeraj Macha1 1
Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA, 2Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany, and Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim, Germany
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Abstract
Keywords
1. The absorption, biotransformation and excretion of empagliflozin, an SGLT2 inhibitor, were evaluated in eight healthy subjects following a single 50 mg oral dose of empagliflozin containing 100 mCi [14C]-empagliflozin. 2. Radioactivity was rapidly absorbed, with plasma levels peaking 1 h post-dose. Total exposure was lower in blood versus plasma, consistent with moderate (28.6–36.8%) red blood cell partitioning. Protein binding was 80.3–86.2%. 3. Most of the radioactive dose was recovered in urine (54.4%) and faeces (41.1%). Unchanged empagliflozin was the most abundant drug-related component in plasma, representing 75.5–77.4% of plasma radioactivity and 79.6% plasma radioactivity AUC0–12 h. Unchanged empagliflozin was the most abundant drug-related component in urine and faeces, representing 43.5% (23.7% of dose) and 82.9% (34.1% of dose) of radioactivity in urine and faeces, respectively. Six metabolites were identified in plasma: three glucuronide conjugates representing 4.7–7.1% of AUC0–12 h and three less abundant metabolites (50.2–1.9% AUC0–12 h). The most abundant metabolites in urine were two glucuronide conjugates (7.8–13.2% of dose) and in faeces was a tetrahydrofuran ring-opened carboxylic acid metabolite (1.9% of dose). 4. To conclude, empagliflozin was rapidly absorbed and excreted primarily unchanged in urine and faeces. Unchanged parent was the major drug-related component in plasma. Metabolism was primarily via glucuronide conjugation.
Absorption, excretion, metabolic pathway, metabolite, pharmacokinetics, radiolabeled
Introduction Type 2 diabetes mellitus (T2DM) is a chronic disease characterised by hyperglycaemia and progressive beta-cell failure (DeFronzo, 2009). The prevalence and incidence of T2DM are increasing worldwide (International Diabetes Federation, 2013). In the US and Europe, T2DM is a leading cause of cardiovascular disorders, blindness, end-stage renal disease and amputations (Inzucchi et al., 2012). The ultimate aim of managing patients with T2DM is to control glycaemia and thus prevent or delay the development of complications (Inzucchi et al., 2012). However, despite the availability of many anti-diabetes agents, many patients fail to achieve recommended glycaemic targets (Esposito et al., 2012). The sodium glucose cotransporter 2 (SGLT2), located in the proximal tubule, is the transporter primarily responsible
Address for correspondence: Lin-Zhi Chen, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA. Tel: + 1 (203) 7787870. E-mail: [email protected]
History Received 21 October 2014 Revised 10 December 2014 Accepted 12 December 2014 Published online 30 December 2014
for reabsorption of the glucose filtered by the kidney (DeFronzo et al., 2012). In healthy individuals, virtually all of the 180 g/d of glucose filtered by the kidney is reabsorbed into the bloodstream (Gerich, 2010). Inhibition of SGLT2 reduces renal glucose reabsorption and increases urinary glucose excretion (UGE), thereby reducing hyperglycaemia in patients with T2DM (DeFronzo et al., 2012). Empagliflozin is a potent and selective SGLT2 inhibitor (Grempler et al., 2012) developed as a treatment for T2DM. The pharmacokinetics of empagliflozin are similar in healthy subjects and patients with T2DM (Heise et al., 2013a,b; Seman et al., 2013). After oral administration of an empagliflozin tablet, empagliflozin is rapidly absorbed, with peak plasma concentrations occurring a median 1.5 h postdose (Heise et al., 2013a). Thereafter, plasma concentrations decline in a biphasic manner with a rapid distribution phase and a relatively slow terminal phase. The single-dose and steady-state pharmacokinetics of empagliflozin are similar, suggesting linear pharmacokinetics with respect to time; systemic exposure to empagliflozin increases in a doseproportional manner (Heise et al., 2013a,b).
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Empagliflozin provides a sustained increase in UGE in patients with T2DM (Heise et al., 2013a). In phase III trials in patients with T2DM, empagliflozin, given as monotherapy or as add-on to other anti-diabetes therapies, was consistently shown to improve glycaemic control, to reduce body weight and blood pressure, and to be well-tolerated, with a low risk of hypoglycaemia (Barnett et al., 2014; Haering et al., 2013, 2014; Kovacs et al., 2014; Roden et al., 2013). The current study was undertaken to determine the metabolism, pharmacokinetics and excretion of a single oral dose of [14C]-empagliflozin in healthy subjects.
Materials and methods
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Subjects Eight healthy male non-smokers were enrolled. Inclusion criteria included age 18–55 years and body mass index (BMI) of 18.5–29.9 kg/m2. Exclusion criteria included medical or laboratory findings of clinical relevance, Gilbert’s syndrome, irregular defecation pattern (51 bowel movement/day); history of gastrointestinal surgery (except appendectomy), orthostatic hypotension, fainting, blackouts, hypersensitivity, habitual tobacco/nicotine use (within prior 6 months) or alcohol abuse; blood donation within 4 weeks of, or during, the trial; participation in more than one study with a radiolabelled investigational drug in the last year; or administration of a radiolabelled drug within the last 6 months. The use of prescription or over-the-counter drugs and herbal preparations was restricted within 14 d of study drug administration, as was the use of drugs with a half-life >24 h within 1 month. All subjects provided written informed consent prior to participation. Study design This was a phase I, open-label, single-centre, single-dose study. Subjects received 50 mg empagliflozin containing [14C]-radiolabelled empagliflozin in solution following an overnight fast of 10 h. The empagliflozin solution was administered via syringe directly into the mouth, after which the subject drank 240-mL water. The dose of empagliflozin administered to each subject was calculated based on the difference in the weight of the syringe before and after dose administration. Fasting continued for 4 h after dosing. Standard meals were served 4.5, 8 and 10.5 h after study drug administration. There were no special requirements regarding food intake on other study days. Subjects remained in the study centre for 7 and 15 d after drug administration; they were discharged when 90% of the administered radioactivity had been recovered in the urine and faeces or 1% of the administered radioactivity was recovered in total in the urine and faeces over two consecutive 24-h periods. The study protocol was approved by the local Institutional Review Board and complied with the Declaration of Helsinki in accordance with the International Conference on Harmonisation Harmonised Tripartite Guideline for Good Clinical Practice. Test compound and chemicals Empagliflozin powder for oral solution and [14C]-empagliflozin powder were manufactured by Boehringer Ingelheim
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Pharmaceuticals, Inc. (Ridgefield, CT). [14C]-empagliflozin material had a radiopurity of >97%, as determined by highperformance liquid chromatography (HPLC) coupled with flow scintillation detection. A mixture of empagliflozin and [14C]-empagliflozin was reconstituted with polyethylene glycol 400/water (60/40, w/w) at a concentration of 5 mg/g to provide a radioactive dose of 100 mCi per subject (49.1 mg empagliflozin plus 0.9 mg [14C]-empagliflozin, adjusted according to the specific activity). The specific activity of [14C]-empagliflozin was 2.13 mCi/mg. Three empagliflozin glucuronide standards (6-O-glucuronide, 2-O-glucuronide and 3-O-glucuronide conjugates) were synthesised by Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT). The liquid scintillation cocktails Ultima Gold, Ultima FLO-MÕ and the absorbent Carbo-Sorb were obtained from PerkinElmer (Meriden, CT). All other chemicals and solvents were of HPLC or analytical grades and obtained from reliable commercial sources. Sample collection Blood samples were collected at the following intervals relative to study drug administration for pharmacokinetic analyses of empagliflozin in plasma, and radioactivity in blood and plasma: pre-dose, 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 h. Metabolite profiling and haematocrit (for evaluation of RBC partitioning: Cblood cells/Cplasma ratio of radioactivity) were conducted on samples collected 1 h prior to dose (for haematocrit only) and 2, 6 and 12 h post-dose. Plasma protein binding was determined using samples collected before (1 h) and 1.5 and 3 h after study drug administration. Urine samples were collected to determine the concentrations of empagliflozin and radioactivity in urine, and to investigate the metabolic profile of empagliflozin. A blank urine sample was collected 2 h prior to study drug administration. Urine was collected during the following intervals after study drug administration: 0–4, 4–8, 8–12, 12–24, 24–28, 28–32, 32–36 h and then in 24-h intervals until the end of the study. A faecal sample was collected within 24 h prior to study drug administration, if possible. All faeces were then collected over 24-h time intervals up to 168 h post-dose for the analysis of radioactivity and metabolic profiling. The weight of the samples was recorded. Safety assessments Safety was assessed through monitoring of vital signs (blood pressure and pulse rate), 12-lead electrocardiogram (ECG), clinical laboratory tests and adverse events (AEs) and via a global tolerability assessment (good, satisfactory, not satisfactory, or bad) undertaken by the investigator at discharge. Bioanalytical methods Empagliflozin in plasma and urine Validated HPLC methods with tandem mass spectrometric (MS/MS) detection were used to determine empagliflozin concentrations in plasma and urine using K3EDTA as an
DOI: 10.3109/00498254.2014.999141
anti-coagulant. Empagliflozin and the internal standard, [13C6]-empagliflozin, were extracted from plasma and urine using solid-supported liquid extraction. After evaporation under nitrogen, the residue was reconstituted and analysed using liquid chromatography (LC) with MS/MS. The LC/ MS/MS method quantitated only the unlabelled portion of the drug. As unlabelled drug accounted for >99% of the total dose, the concentrations of unlabelled drug were treated as the total drug concentrations, with no correction for the labelled drug. The lower limit of quantification for empagliflozin in plasma and urine was 1.11 nM and 4.44 nM, respectively, with linearity to 1110 nM and 4440 nM, respectively.
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Radioactivity measurement Whole blood, plasma, urine and faeces were analysed for radioactivity by liquid scintillation counting (LSC). Each sample was homogenised prior to radioanalysis. Faecal samples were diluted in water prior to homogenisation. Plasma and urine samples were analysed directly; blood and faecal samples were combusted (Model 307 Sample Oxidizer, Perkin Elmer, Waltham, MA), and the resulting 14CO2 trapped in a mixture of Perma Fluor and Carbo-Sorb prior to LSC. All samples were analysed using Model 2900T LSC (Perkin Elmer, Waltham, MA) using Ultima Gold XR scintillation cocktail for 5 min or 100 000 counts. All samples were analysed in duplicate. Pharmacokinetic analysis Non-compartmental pharmacokinetic parameters for empagliflozin and total radioactivity were determined using WinNonLinTM software (version 5.2, Pharsight Corporation, Sunnyvale, CA). Maximum plasma concentrations (Cmax) and time to Cmax (tmax) values were determined directly from the plasma concentration–time curves. The terminal half-lives (t1/2) were calculated as the quotient of ln(2) and the apparent terminal rate constant (lz), where lz was estimated from a regression of ln(C) versus time over the terminal loglinear disposition portion of the concentration-time profiles. AUC0–1 was estimated as the sum of the area under the curve (AUC) over the time interval 0 to the time of the last quantifiable data point with the extrapolated area given by the quotient of the last measured concentration and lz. The fraction of dose excreted in the urine (feurine) was calculated as the quotient of the sum of drug/radioactivity excreted in the urine over all observed intervals and the dose administered, and the fraction of the dose of total radioactivity excreted in the faeces (fefaeces) was calculated in a similar way. Plasma protein binding of radioactivity was determined using equilibrium dialysis for 6 h at 37 C. The ratio of concentration of total radioactivity in blood cells and plasma (Cblood cells/Cplasma) was calculated according to the following formula, where Cblood cells is the concentration in blood cells, Cplasma is the concentration in plasma, and HC is the haematocrit (decimal; determined using standard laboratory procedures): Cblood cells =Cplasma ¼
ðCblood cells Cplasma Þ ð1 HCÞ : HC Cplasma
Biotransformation and mass balance of empagliflozin
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Metabolite sample extraction The 2-h and 6-h plasma samples were profiled individually. Due to low plasma radioactivity, the 12-h samples from all subjects were combined. An equal volume of water was added to every sample. The diluted samples were subjected to solidphase extraction (SPE) with preconditioned Oasis MCX SPE cartridges (35 cc/6 g for the 2-h and 12-h samples; 20 cc/1 g for 6-h samples; Waters, Milford, MA). After loading, the SPE cartridges were washed sequentially with 0.5% acetic acid, water and 5% methanol in water and the sample was eluted with methanol. The eluate was evaporated to 0.2 mL under a nitrogen stream at 35 C (Zymark Turbovap LV evaporator, GenTech Scientific Inc., Hopinton, MA). The final samples were reconstituted in methanol and water (50:50) and transferred to autosampler vials. The overall recovery of radioactivity from plasma was 85.3–99.8%. Urine collected until 48-h post-dose was pooled for each subject; this sample represented 94.4–98.4% of the total radioactivity ultimately recovered in urine. A 100 mL aliquot of pooled urine was extracted (Oasis MCX SPE cartridge 35 cc/6 g; Waters, Milford, MA), washed, eluted, dried (to 0.1 mL) and reconstituted as for plasma samples. The overall recovery of radioactivity from urine was 87.8–100%. Faeces collected until 144-h post-dose was pooled for each subject. This represented 94.2–99.8% of total radioactivity ultimately recovered in faeces. A 10 mL aliquot of the pooled faecal homogenate was centrifuged at 10 000 rpm for 15 min at 10 C. The supernatant was separated and the pellet extracted with methanol (2) and 10% acetic acid in methanol. The extracts were combined and then evaporated to dryness under a nitrogen stream at 35 C. The residue was sonicated to dissolve in 20 mL water and then combined with the supernatant. The combined sample was extracted (Oasis MCX SPE cartridges 35 cc/6 g; Waters, Milford, MA), washed, eluted, dried (to 0.1–0.2 mL) and reconstituted (with 1 mL methanol in water, 50:50) as for plasma and urine samples. The overall extraction recovery from faeces was 82.7–100%. Metabolite profiling and identification Metabolite profiling and identification in plasma, urine and faeces were conducted using an LC/radiochromatography/ MS/MS system. Empagliflozin metabolites were identified based on HPLC retention time, radiochromatography and MS. MS, MS/MS, triple mass spectrometry (MS3) and accurate mass measurements were performed for metabolite structure elucidation. The system comprised an Agilent 1200 HPLC system (Palo Alto, CA), with a Thermo LTQ Orbitrap XL mass spectrometer (San Jose, CA) and a PerkinElmer 625 TR flow scintillation analyser (Shelton, CT) serving as detectors. Radiochromatograms generated from the flow scintillation analyser were processed using ProFSA software (PerkinElmer, Shelton, CT). The LTQ Orbitrap mass spectrometer was operated with Xcalibur 2.0 software (Thermo Scientific, San Jose, CA, USA). Thermo MetWorks 1.1 software (Thermo Scientific, San Jose, CA) with mass defect filter was used to assist metabolite identification. Authentic standards for empagliflozin and its three glucuronide conjugates were used for confirmation of identification.
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Results
Radioactivity in blood and plasma
Subjects
The shapes of the profiles for radioactivity in blood and plasma were similar (Figure 2). Radioactivity was rapidly absorbed, reaching peak plasma levels 1 h after administration, after which plasma concentrations declined in a biphasic manner with a rapid distribution phase and a slower elimination phase. Total radioactivity levels were below the limit of quantification at 36 h post-dose in the blood and at 48 h post-dose in the plasma. Radioactivity exposure was
Eight subjects aged 21–53 years with a mean BMI of 25.0 kg/m2 (range 21.0–28.9 kg/m2) and a mean body weight of 79 kg were enrolled and received study drug. One or more AE was reported in five subjects. All AEs were mild in intensity. Gastrointestinal AEs (abdominal distension, flatulence) experienced by three subjects were considered drug-related by the investigator. The investigator considered the global tolerability of empagliflozin to be good for all eight subjects. Administration of empagliflozin had no effect on vital signs or ECG and no consistent or clinically relevant effect on clinical laboratory findings.
Table 1. Pharmacokinetics of radioactivity and empagliflozin after a single dose of empagliflozin 50 mg containing 100 mCi [14C]-empagliflozin in eight healthy subjects. Radioactivity Blood (n ¼ 8)
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Mass balance The overall recovery of radioactivity in the urine and faeces was 95.6%(range: 93.0–99.4%) over 168 h after administration of study drug. Most (94.3%) of the radioactivity was recovered in the first 120 h. Figure 1 shows the cumulative amounts of radioactivity excreted in the urine and faeces as a percentage of the dose and in nM. A mean of 54.4% of the radioactive dose was excreted in the urine and 41.2% in the faeces (Figure 1; Table 1). Figure 1. Cumulative amounts of radioactivity excreted in the urine and faeces (a) as % of dose and (b) as nmol following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin to eight healthy subjects.
Plasma (n ¼ 8)
Empagliflozin Plasma (n ¼ 8)
16 100 ± 20.9 15 600 ± 19.5 AUC0–1 (nmolh/L) 11 000 ± 19.5 Cmax (nmol/L) 1470 ± 21.3 2200 ± 21.4 2260 ± 20.9 tmax (h) 1.0 (0.8–1.5) 1.0 (0.8–1.0) 0.9 (0.8–1.0) t½ (h) 8.7 ± 12.8 9.2 ± 6.9 15.9 ± 46.3 CLR,0–tz (mL/min) 63.7 ± 21.2 34.3 ± 27.9 feurine,0–tz (%) 54.4 ± 6.4 28.6 ± 13.6 fefaeces,0–tz (%) 41.2 ± 7.2 Not measured Data are mean ± %CV except tmax, which is median (range).
Biotransformation and mass balance of empagliflozin
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Figure 2. Concentration versus time profiles for radioactivity in blood and plasma and empagliflozin in plasma following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin to eight healthy subjects.
Table 2. Mean radioactivity contributions of empagliflozin and its metabolites after administration of a single dose of empagliflozin 50 mg containing 100 mCi [14C]-empagliflozin in eight healthy subjects. % of Sample radioactivity [concentration(nM) in plasma] Compound Empagliflozin M626/3 M626/1 M626/2 M482/1 M464/1 M468/1
2h 77.4 7.4 6.2 3.7 1.2 0.5 0.4
[1320] [127] [109] [62.8] [24.3] [8.5] [5.6]
6h 75.5 6.3 5.0 6.0 1.8 0.4 0.2
[638] [53.4] [42.1] [49.4] [17.1] [3.1] [1.5]
% AUC0–12 h
12 h 76.2 5.4 5.2 3.3 3.1 1.1
[283] [20.1] [19.3] [12.3] [11.5] [4.1] –
% of Sample radioactivity [% dose]
nM h/L
% Total
Faeces
7999 708.3 595.9 472.3 192.9 53.3 24.3
79.6% 7.1% 5.9% 4.7% 1.9% 0.5% 0.2%
82.9 [34.2] – – – 4.6 [1.9] 2.6 [1.1] 1.4 [0.6]
lower in blood than plasma (Figure 2; Table 1), consistent with moderate partitioning of radioactivity into RBC. Mean RBC:plasma concentration ratios of radioactivity were 28.6%, 30.2% and 36.8% at 2, 6 and 12 h post dose. Protein binding of radioactivity varied little across time points and ranged from 80.3–86.2%. Empagliflozin in plasma and urine The rates of absorption and disposition of total radioactivity and empagliflozin were similar (Figure 2; Table 1). There were no major differences in the pharmacokinetics of total radioactivity and empagliflozin (Table 1). When empagliflozin and radioactivity exposure were compared, the majority of total radioactivity in plasma appears to be unchanged empagliflozin. A total of 28.6% of the empagliflozin dose was excreted unchanged in the urine. Metabolite profiles Unchanged empagliflozin was the most abundant drug-related component in the plasma, urine and faeces (Table 2; Figures 3a–c). No major metabolites were detected in plasma. Empagliflozin accounted for 75.5–77.4% of plasma radioactivity or 79.6% of plasma radioactivity AUC0–12 h. Six minor metabolites were identified in plasma. Three glucuronide conjugates (M626/1, M626/2, M626/3) were identified using authentic standards (Figure 3a) and
Urine 43.5 24.1 14.4 3.9 5.2 1.5
[23.7] [13.2] [7.8] [2.1] [2.8] [0.9] –
Total % dose (urine + faeces) 57.9 13.2 7.8 2.1 4.7 2.0 0.6
represented 3.7–7.4% of plasma radioactivity and 4.7–7.1% of AUC0–12 h. Three less abundant metabolites were tentatively identified and represented 51.9% of AUC0–12 h: a tetrahydrofuran ring-opened carboxylic acid metabolite (M482/1), an oxidation/dehydrogenation metabolite (M464/1) and a tetrahydrofuran ring-opened dihydroxyl metabolite (M468/1) (Table 2). The metabolic pathways and associated structures are presented in Figure 4. Urinary excretion accounted for 54.4% of the radioactive dose (Table 1). Empagliflozin accounted for 43.5% of the radioactivity detected in urine (23.7% of the dose). The remaining radioactivity was accounted for primarily by five metabolites, all of which were also observed in the plasma (the three glucuronide conjugates, M482/1 and M464/1) (Table 2, Figure 3b). Faecal excretion accounted for 41.2% of the radioactive dose (Table 1). Empagliflozin accounted for 82.9% of the radioactivity detected in faeces (34.2% of the dose). The remaining faecal radioactivity was accounted for primarily by the three less abundant metabolites, which were also found in the plasma (Table 2, Figure 3c). Metabolite identification Empagliflozin Empagliflozin corresponded to the radioactive peak eluting at 65.2 min in plasma, urine and faeces, as it had the same
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Figure 3. Radiochromatograms of 2-h plasma (a), urine (b) and faeces (c) from subject M107 following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin.
retention time, MS and MS/MS patterns as the authentic standard. The molecular ions at m/z 451 (positive mode) and m/z 449 (negative mode) with an isotope ratio of 0.35 for [M + 2]/M was characteristic of the presence of one chlorine atom. MS/MS produced several abundant fragment ions in positive mode, the majority of which were also observed in negative mode, which showed the loss of the tetrahydrofuran ring, corresponding to the m/z 379 ion (Figure 5).
M464/1 A radioactive peak eluting at 62.1 min was observed in plasma, urine and faeces. Positive MS showed an elemental composition of C23H26O8Cl. MS/MS was consistent with an apparent oxidation/dehydrogenation metabolite (Figure 7). M468/1
Radioactive peaks eluting at 49.5, 54.3 and 60.9 min were found in plasma and urine and had the same retention, molecule weight, elemental composition and MS/MS spectra as respective authentic standards of the three glucuronide conjugates of empagliflozin. They had similar MS/MS results with characteristic empagliflozin fragments at m/z 395, 379, 371, 359 and 329.
A radioactive peak eluting at 56.7 min was observed in plasma and faeces, corresponding to a negative molecular ion at m/z 467, 18 Da more than empagliflozin. High resolution MS indicated that the 18 Da were from one oxygen and two hydrogen atoms. The isotope ratio of [M + 2]/[M] was 0.35, suggesting that the chlorine atom was retained. Negative MS/MS gave a predominant fragment at m/z 379 (Figure 8). MS3 for this fragment was dominated by fragments at m/z 301, 289 and 259. Mass spectral results were consistent with a tetrahydrofuran ring-opened dihydroxyl structure.
M482/1
Discussion
A radioactive peak eluting at 40.4 min was observed in plasma, urine and faeces corresponding to a negative molecular ion at m/z 481, 32 Da more than empagliflozin. The isotope ratio of [M + 2]/[M] was 0.35, suggesting that the chlorine atom was retained. High resolution MS yielded the most likely formula, C23H28O9Cl. Negative MS/MS gave fragments at m/z 379, 361, 301, 319 and 259, suggesting that the 32 Da were added to the furan ring (Figure 6). Based on the mass spectral analyses, M482/1 was tentatively identified as a tetrahydrofuran ring-opened carboxylic metabolite.
Empagliflozin was rapidly absorbed, underwent limited metabolism and was excreted primarily unchanged in the urine and faeces. The most abundant metabolites in plasma were three glucuronide conjugates (2-O, 3-O and 6-O glucuronide). Systemic exposure of each metabolite was less than 10% of total drug-related material. In addition, the PK profile including AUC0–1, Cmax, tmax and t1/2 was similar between empagliflozin and total plasma radioactivity, indicating minimal metabolite accumulation at steady state upon multiple dosing. There were no major human metabolites that
M6261/1, M626/2 and M626/3
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DOI: 10.3109/00498254.2014.999141
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Figure 4. Metabolic pathway for empagliflozin following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin to healthy subjects.
required non-clinical safety assessments (U.S. Department of Health and Human Services, 2008). Similar to plasma, unchanged empagliflozin was the major drug-related component in excreta and accounted for 57.9% of the dose in urine and faeces combined. The major elimination pathway for empagliflozin was via the urine and it is possible that active secretion could account for a portion of the empagliflozin excreted unchanged in the urine. Empagliflozin is a substrate of the human organic anion transporter 3 (OAT3), which is expressed on the basolateral membrane of the renal proximal tubule and is involved in the renal excretion of many drugs (data on file). Thus, OAT3 could be responsible for the active secretion of empagliflozin into the urine. The biotransformation of empagliflozin in humans mainly involved glucuronide conjugation and, to a lesser extent, oxidation. The glucuronidation took place on the glucose moiety to produce metabolites M626/1, M626/2 and M626/3. Oxidation on the tetrahydrofuran ring followed by ring opening led to the formation of dihydroxyl metabolite M468/1 and carboxylic acid metabolite M482/1, possibly via an aldehyde intermediate (not detected). Oxidation was also responsible for the formation of M464/1. All these empagliflozin metabolites have also been seen in rats, mice or dogs (data on file). Compared to humans, metabolic
pathways involving oxidation were much more prominent in the animals and several other oxidation metabolites were found in the animals. The biotransformation of empagliflozin is different to that of the SGLT2 inhibitors canagliflozin and dapagliflozin. While unchanged canagliflozin is the most abundant drugrelated component in plasma, representing 60% of plasma radioactivity, it has two major metabolites, both glucuronide conjugates, which represent 19% and 16% of radioactivity in plasma (Mamidi et al., 2014). Canagliflozin is primarily excreted in the faeces, with 60% of the radioactive dose in faeces and 33% in urine. Unchanged canagliflozin is the primary drug-related component in faeces, representing 39% of the radioactive dose. Unchanged canagliflozin is not present in the urine, only the metabolites which represent 14% and 18% of the radioactive dose (Mamidi et al., 2014). In contrast to empagliflozin and canagliflozin, dapagliflozin is extensively metabolised. A ratio of dapagliflozin concentration to total plasma radioactivity of 0.3 suggests that metabolites account for a significant proportion of drug-related components in plasma (Obermeier et al., 2010). A major metabolite, dapagliflozin 3-O-glucuronide, is found at similar concentrations to dapagliflozin in plasma; together these account for >72% of plasma
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Figure 5. Positive MS/MS of empagliflozin [M + H]+ ion at m/z 451 (a) and negative MS/MS of empagliflozin [M H] ion at m/z 449 (b) from authentic standard.
Figure 6. Negative MS/MS of M482/1 molecular ion at m/z 481 from the urine of subject M115 following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin.
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Figure 7. Positive MS/MS of M464/1 molecular ion at m/z 465 from the faeces of subject M107 following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin.
Figure 8. Negative MS/MS of M468/1 molecular ion at m/z 467 from the faeces of subject M115 following administration of a single 50 mg dose containing 100 mCi [14C]-empagliflozin.
radioactivity (Obermeier et al., 2010). The formation of dapagliflozin 3-O-glucuronide is mediated by uridine diphosphate glucuronosyltransferase (UGT) 1A9, an enzyme present in the liver and kidney, with CYP-mediated metabolism as a minor clearance pathway (Bristol-Myers Squibb/AstraZeneca, 2014). Dapagliflozin is primarily excreted in the urine, with 75% of the radioactive dose excreted in urine and 21% in faeces (Bristol-Myers Squibb/ AstraZeneca, 2014; Obermeier et al., 2010). Unchanged dapagliflozin represents 1.6% of the radioactive dose in the urine and 15% in the faeces (Bristol-Myers Squibb/ AstraZeneca, 2014; Obermeier et al., 2010). The results of this study demonstrated that the major elimination pathway for empagliflozin is via the urine. In light of this, a study was undertaken to determine the effect of renal impairment on the pharmacokinetics of a single 50 mg dose of empagliflozin. This study found that there were no clinically relevant increases in exposure in patients
with renal impairment compared to patients with normal renal function, suggesting that no dose adjustments of empagliflozin are required in patients with renal impairment (Macha et al., 2014a). Similarly, a hepatic impairment study conducted to investigate whether decreased hepatic metabolism of empagliflozin might increase exposure found that hepatic impairment had no clinically relevant effects on drug exposure, suggesting that no dose adjustment of empagliflozin is required in patients with hepatic impairment (Macha et al., 2014b).
Conclusion Following a single oral dose in healthy subjects [14C]empagliflozin was rapidly absorbed and excreted primarily unchanged in the urine and faeces. A total of 54.4% of the empagliflozin dose was excreted in urine and 41.2% in the faeces. Empagliflozin underwent limited metabolism, as
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demonstrated by unchanged parent being the predominant drug-related component in the plasma, urine and faeces. Six minor metabolites (each less than 10% of total drug-related material) were identified. Radioactivity exposure was lower in blood than plasma, consistent with moderate partitioning into RBC.
Acknowledgements The authors acknowledge the contribution of Debra Mandarino, MD, who was the principal investigator at the clinical site (Covance Laboratories, Madison, WI).
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Declaration of interest Medical writing assistance, supported financially by Boehringer Ingelheim, was provided by Clare Ryles and Wendy Morris of Fleishman-Hillard Group, Ltd, during the preparation of this article. This study was funded by Boehringer Ingelheim. All authors are employees of Boehringer Ingelheim. The authors were fully responsible for all content and editorial decisions and were involved at all stages of article development and have approved the final version.
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