Pharmacokinetics , Biotransformation , Distribution and Excretion of Empagli-flozin , a Sodium-Glucose CoTransporter ( SGLT 2 ) Inhibitor , in Mice , Rats , and Dogs

Introduction Pharmacokinetics, Biotransformation, Distribution and Excretion of Empagliflozin, a Sodium-Glucose Co-Transporter (SGLT 2) Inhibitor, in Mice, Rats, and Dogs Chen L*1, Mao Y1, Sharp DE1, Schadt S2,5, Pagels S1, Press R3, Cheng T3, Potchoiba MJ3 and Collins W4 1Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA 2Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany 3Covance Laboratories, Inc., Madison, WI, USA 4XenoBiotic Laboratories, Inc., Plainsboro, NJ, USA 5F. Hoffmann-La Roche Ltd, Basel, Switzerland *Corresponding author: Chen L, Ph.D., Drug Metabolism and Pharmacokinetics US, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT, USA 06877, Fax: 203-791-6003, Tel: 203-778-7870, E-mail: Lin_zhi.chen@boehringer-ingelheim.com Citation: Chen L, Mao Y, Sharp DE, Schadt S, Pagels S, et al. (2015) Pharmacokinetics, Biotransformation, Distribution and Excretion of Empagliflozin, a Sodium-Glucose Co-Transporter (SGLT 2) Inhibitor, in Mice, Rats, and Dogs. J Pharm Drug Devel 3(3): 302. doi: 10.15744/2348-9782.3.302


Animals
The mass balance studies were conducted in male and female mice and rats and the doses were administered as solutions.Details on the formulations and doses administered are provided in the supplementary material.Each animal received a single [ 14 C]empagliflozin dose by intravenous (IV) (5 mg/kg for mice and 0.5 mg/kg for rats) or per oral (PO) (250 mg/kg for mice and 5 mg/kg for rats) at a dose level based upon individual body weights taken prior to dosing.The actual amount of dose formulation administered to each animal was determined by weighing the loaded dose syringe before dose administration and the emptied syringe after dose administration.

Mouse and rat mass balance studies
Urine was collected on dry ice at 24-h intervals for 168 h.Feces were collected on dry ice (mice and rats) at 24-h intervals for 168 h.Cages were rinsed at 24-h intervals post-dose.
Bile was obtained from male rats after PO dose and collected on dry ice hourly for 8 h, and then from 8-24 and 24-48 h postdose.
Pharmacokinetic studies were conducted in CD-1 mice and Wistar Han rats after a single PO or IV dose of [ 14 C]-empagliflozin ( 14 C-PK) or unlabeled empagliflozin (cold PK).The rat PK studies with unlabeled empagliflozin were conducted only in male animals as per our normal procedure.Blood was collected from 4 mice/sex/time point in tubes containing K 3 EDTA as the anticoagulant from the retro-orbital venous plexus at 0.33, 1, 2, 4, 6, 8, and 24 h after dosing.Blood was collected from rats via a jugular vein cannula with K 3 EDTA as the anticoagulant at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after dosing.Blood obtained via cardiac puncture were collected from separate mice and rats for metabolite identification.Plasma samples were prepared by centrifugation of the whole blood.Plasma samples were stored at approximately -20 o C prior to analysis.

Mouse and rat PK studies
The PK studies with unlabeled empagliflozin were conducted only in male dogs as per our normal procedure.The radiolabeled studies were conducted in dogs of both sexes.The volume of radiolabeled dose formulation administered to each dog was calculated based on the body weight taken on the day of dosing.The actual amount administered was determined by weighing the dose syringe before and after dose administration.The IV dose was administered via a cephalic vein and the oral dose was administered via oral gavage.

QWBA study
The Quantitative Whole-Body Autoradiography (QWBA) study was conducted in male rats following a single PO dose of [ 14 C]empagliflozin.Each animal received a single [ 14 C]-empagliflozin dose by oral gavage at a dose level based upon individual body weights taken prior to dosing.Rats were euthanized by an overdose of isoflurane anesthesia.The carcasses were frozen in a hexane dry ice bath and stored at -20 o C. The carcasses were embedded in carboxymethyl cellulose blocks.Blood samples fortified with [ 14 C]-empagliflozin were placed in to the frozen block to serve as standards.Sagittal sections (40 µm thick) at five levels of interest were prepared in a Leica CM 3600 cryomicrotome.Mounted sections were wrapped with Mylar film and exposed to phosphorimaging screens for 4 days.Exposed screens were scanned using an Amersham Biosciences Storm.Tissue concentrations were interpolated from the standard curve.
PK parameters were calculated from blood and plasma radioactivity concentration data using WinNonlin Professional Edition, Version 5.2 (Certara Corporation, St. Louis MO) for dogs and Kinetica Version 4.4.1 (Thermo Scientific, Philadelphia, PA) for rats and mice.PK parameters calculated included half-life (t 1/2 ), area under the concentration-time curve from time 0 to the last measurable time point (AUC 0-t ), and area under the concentration-time curve from 0 to infinity (AUC 0-∞ ).A concentration of "0" µM was assumed for the predose concentrations as well as for those samples with levels below the lower limit of quantitation.
Homogenate of feces collected up through 48 h (mice and dogs) or 72 h (rats) were pooled at equal percentages (by weight) according to gender, and this represented ≥ 97.3% of total radioactivity excreted in feces from mice, rats or dogs.The pooled fecal homogenate was centrifuged, and the resultant pellet was extracted with methanol, acetonitrile and 10% acetic acid in methanol.The extracts were combined with the supernatant and concentrated at 37 o C. For mice, the concentrated samples were centrifuged prior to analysis.For rats and dogs, the concentrated samples were diluted with water and then extracted with SPE following the procedure used for plasma extraction.The overall extraction recovery of radioactivity from feces was ≥ 89.5%.
Rat bile was collected up to 48 hours post-dose from 4 male rats after single oral administration of [ 14 C]-BI 10773 (5 mg/kg).For metabolite profiling, all available 2 to 48-hr bile samples were combined, diluted with methanol at a ratio of 7 : 3 (bile : methanol; v:v) and centrifuged, and resulting supernatant was separated for analysis.

PK analysis
Analysis of Radioactivity: Feces were homogenized in water (mouse and rat) or 20: 80 ethanol: water (dog).Fecal homogenates for rat and dog were combusted in a Packard 307 Sample Oxidizer (Perkin Elmer, Waltham, MA, USA) and the resulting [ 14 C]-CO 2 was trapped in Carbo-Sorb.Mouse fecal homogenates were combusted in a Harvey Biological Sample oxidizer and the resulting [ 14 C]-CO 2 was trapped in Harvey Scintillation.Levels of radioactivity in plasma, urine, bile, cage rinse/wash and CO 2 -trap samples were analyzed by LSC (Models Tricarb 2900 Perkin Elmer, Waltham, MA, USA).Scintillation counting data expressed in counts/ min (cpm) were automatically corrected for counting efficiency using the external standardization technique and an instrument stored quench curve.

Sample preparation and analysis
Metabolite Profiling/id: Mouse, rat and dog plasma samples collected at different time points following oral administration of [ 14 C]-empagliflozin were pooled according to gender and time point.The pooled plasma samples were diluted with water and then extracted using Waters Oasis MCX 20 cc/1 g solid phase extraction (SPE) cartridges (Waters, Milford, MA).After loading, the SPE cartridge was washed with 0.5% acetic acid or 0.1 N HCl, water and 5% methanol in water, and eluted with methanol.The eluate was concentrated under 37 o C with a Zymark Turbovap LV (GenTech Scientific, Inc.New York, NY) or similar evaporator, and reconstituted in methanol and water for analysis.The overall extraction recovery of radioactivity from plasma was ≥ 87.1%.Urine collected from 0-24 h (mouse), 0-120 h (rats) or 0-24 h period (dogs) was pooled at equal percentages (by volume) according to gender; this represented ≥ 93.9% of the total radioactivity excreted in urine from rats, mice or dogs.For analysis, the pooled urine was prepared by centrifugation (mice) or by SPE (rats and dogs) following the similar SPE procedures for plasma.The overall extraction recovery of radioactivity from urine was ≥ 95.9%.Metabolite profiling and identification was carried out with an LC/radio-detection/MS system.The system consisted of an Agilent 1100 or 1200 HPLC system (Agilent, Palo Alto, CA) with a LTQ Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) and a flow scintillation analyzer serving as detectors.An ARCTM (AIM Research Co., Hockessin, DE), PerkinElmer 625 TR (PerkinElmer, Shelton, CT) or Radioflow detector LB 509 (Berthold, Bad Wildbad, Germany) was used as the flow scintillation analyzer.The MS and flow scintillation analyzers functioned in parallel by splitting the 0.6 mL/min flow from the HPLC column in a 1:20 (or 1:10 for mouse) ratio, with one part going to the mass spectrometry and 20 (or 10) parts to the flow scintillation analyzer.

Volume 3 | Issue 3
Metabolite separation was achieved using a reversed-phase HPLC column (Atlantis TM dC18, 150 x 3.9 mm, 3µm; Waters, Milford, MA).Mobile phase A comprised of 90:5:5 H 2 O: MeOH: 100 mM ammonium acetate and mobile phase B was 95:5 MeOH: 100 mM ammonium acetate.The gradient conditions were from 100% to 80% A over 20 min, to 55% A over 30 min, to 20% A over 20 min, and then to 0% A and hold for 8 min at 0.6 mL/min.
Summaries of the pharmacokinetic parameters of plasma empagliflozin and total radioactivity in mice, rats, and dogs are found in the supplemental material.After IV dosing of 5 mg/kg to mice, the mean AUC 0-∞ of plasma total radioactivity was 4.61 and 5.60 µM.h in male and female mice, respectively.The terminal t 1/2 was short, at 1.26 h for males and 0.65 h for females.The mean residence times (MRT) were 0.49 and 0.43 h, in males and females, respectively.After oral dosing at 250 mg/kg to mice, empagliflozin was rapidly absorbed with a t max of 0.67 h and 0.33 h in males and females, respectively.C max values were 97.7 µM and 91.5 µM in male and female mice, respectively.AUC 0-∞ values were 207 µMh and 273 µMh in males and females, respectively.Terminal t 1/2 was long, at 5.59 h for males and 4.31 h for females.The bioavailability of empagliflozin was 89.8% and 96.7%, respectively, in male and female mice.
Fraction collection followed by offline radioactivity counting was used for mouse 8-h plasma due to the low radioactivity in the samples.The instrument setup was the same as the online LC/radiometric/MS system, except that the flow scintillation detector was replaced with a Gilson 204 fraction collector (Gilson, Middleton, WI).The LC fractions were collected in 96-well Scintiplates or Deepwell LumaPlates TM (PerkinElmer, Shelton, CT) which had solid scintillant embedded.The plates were dried using a miVac modular concentrator at 37 o C and then counted with a MicroBeta Trilux 1450 LSC and Luminescence counter or a TopCount NXT counter (PerkinElmer, Shelton, CT).Radiochromatograms were reconstructed using ARC TM Data System (AIM Research Co., Hockessin, DE) or FractionLynx software (Waters, Milford, MA).

PK of empagliflozin
After IV dosing of 0.5 mg/kg to male rats, the mean AUC 0-∞ of empagliflozin was 1.32 µMh.The terminal t 1/2 was moderate at 3.64 h.The MRT was 0.83 h.After oral dosing at 5 mg to male rats, the median t max of empagliflozin occurred at 1 h.The C max and AUC 0- ∞ values were 0.724 µM and 3.99 µM•h, respectively.The terminal t 1/2 was long, at 6.32 h.The bioavailability of empagliflozin was 31.0% in male rats.
After IV dosing of 0.5 mg/kg to male dogs, the mean AUC 0-∞ empagliflozin was 10.6 µM•h.The terminal t 1/2 was moderate at 6.27 h and the MRT was 5.32 h.The clearance value was 1.77 mL/min/kg and the Vss value was 0.568 L/kg.After oral dosing of 5 mg/ kg to dogs, the t max of total radioactivity occurred at 1 h.C max was 17.3 µM.The AUC 0-∞ value was 93.7 µM•h.The terminal half-life was moderate with a t 1/2 of 6.25 h.The bioavailability of empagliflozin was 89.0% in male dogs.After IV dosing of 5 mg/kg to mice, the mean AUC 0-∞ of plasma total radioactivity was 11.1 and 9.51 µM•h in male and female mice, respectively.The terminal t 1/2 was short, at 0.98 h for males and 0.95 h for females.The MRT were 1.26 and 0.93 h, in males and females respectively.After oral dosing at 250 mg/kg to mice, plasma total radioactivity was rapidly absorbed with a t max of 1 h and 0.33 h in males and females, respectively.C max values were 121 µM and 117 µM in male and female mice, respectively.AUC 0-∞ values were 452 µM•h and 386 µM•h in males and females, respectively.Terminal t 1/2 was long, at 7.89 h for males and 12 h for females.

PK of total radioactivity
After IV dosing of 0.5 mg/kg to rats, the mean AUC 0-∞ of plasma total radioactivity was 1.34 and 1.51 µM•h in male and female rats, respectively.The terminal t 1/2 was short, at 0.67 h for males and 1.10 h for females.The MRT were 0.59 and 0.6 h, in males and females respectively.After oral dosing at 5 mg/kg to rats, the median t max of plasma total radioactivity occurred at 1 h in both sexes.C max values were 1.27 µM and 1.10 µM in male and female rats respectively.AUC 0-∞ values were 4.67 µM•h, and 4.20 µM•h in males and females, respectively.The terminal t 1/2 was moderate, at 1.75 h for males and 1.89 h for females.
After IV dosing of 0.5 mg/kg to dogs, the mean AUC 0-∞ of plasma total radioactivity was 10.2 and 10.8 µM•h in male and female dogs, respectively.The terminal t 1/2 was long, at 22.0 h for males and 31.2 h for females.Clearance values were 1.76 and 1.65 mL/ min/kg in males and females, respectively with an overall mean of 1.71 mL/min/kg.In males and females, Vss values were 0.836 and 1.08 L/kg, respectively, and the MRT were 7.87 and 10.9 h, respectively.After oral dosing at 5 mg/kg to dogs, the t max of total radioactivity occurred at 1 h in both males and females.C max values were 16.1 and 15.5µM in male and female dogs, respectively.AUC 0-∞ values were 101 µM•h, and 96.3 µM•h in males and females, respectively.The terminal half-life was moderate with a t 1/2 After oral administration of 250 mg/kg of [ 14 C]-empagliflozin to male and female mice, 36.4% and 48.0% of the radioactive dose was recovered in urine and feces, respectively, from male mice, whereas approximately 44.6% and 40.7% of the radioactive dose was recovered in urine and feces, respectively, from female mice (Table 1).The mean recovery was 96.1 and 95.2 % of dose in males and females, respectively.

Mass balance and excretion
Following a single IV dose of 0.5 mg/kg of [ 14 C]-empagliflozin to male and female rats, the primary route for elimination of drug-derived radioactivity was via feces (Table 1).In males, feces and urine accounted for mean values of 64.4 and 27.5% of the administered dose, respectively.In females, feces and urine represented 61.4 and 28.5% of dose, respectively.The mean overall recoveries in males and females were 92.2 and 90.9% of dose, respectively.After oral administration of 5 mg/kg of [ 14 C]empagliflozin to male and female rats, 27.4% and 28.6% of the total administered radioactive dose was excreted in urine within 168 h (Table 1) in male and female rats, respectively.The respective recovery in the feces was 62.9% and 63.1% of the dose.The total recovery was 92.9% and 92.6% of dose in males and females respectively.There was no sex difference in excretion.
Following a single IV dose of 0.5 mg/kg of [ 14 C]-empagliflozin, the primary route for elimination of drug-derived radioactivity in male and female dogs was via feces (Table 1).In males, feces and urine accounted for mean values of 70.9 and 17.4% of the administered dose, respectively.In females, feces and urine represented 68.6 and 24.6% of dose, respectively.The mean overall recoveries in males and females were 92.4 and 94.7% of dose, respectively.Similar to the IV group, the primary elimination route was via feces following a single oral dose of 5 mg/kg of [ 14 C]-empaglifozin to dogs (Table 1).In males, feces and urine accounted for mean values of 61.4 and 29.6% of the administered dose, respectively.In females, feces and urine represented 69.8 and 19.6% of dose, respectively.The mean overall recoveries in males and females were 93.7 and 91.1% of dose, respectively.
In bile duct-cannulated male rats, 33.3% of the dose was excreted by 48 h post dose (not shown in the table). of 3.60 h in males and 5.16 h in females.The shorter half-life observed in orally dosed animals than in intravenously dosed dogs is due to the lower limit of quantitation found in the intravenously dosed animals, which enabled a larger portion of the terminal elimination phase to be measured.Absorption of drug-derived radioactivity was 102 and 92.1% in males and females, respectively.Metabolite profiling was conducted using plasma from mice, rats and dogs in the oral dose groups only.Plasma samples were obtained at 1, 6, and 24 h for mice, 1 and 8 hours for rats and 1, 4 and 8 hours for dog following a single oral administration of [ 14 C]-empagliflozin at 250 mg/kg (mice) or 5 mg/kg (rats and dogs).These time points represented most of the plasma radioactivity in each species.Relative contributions of empagliflozin and its metabolites to plasma radioactivity are listed in Table 2. To facilitate interspecies comparison, the relative contributions were calculated for the total plasma (AUC) of respective time points for each species.Representative radiochromatogram of 1-h male rat plasma is shown in Figure 2. Empagliflozin was the most abundant drug-related component in all the three species (Table 2), accounting for 40.9% (males) and 93.2% (females) of the plasma radioactivity for mice, 54.3% (males) and 59.3% (females) for rats, and 78.0%(males) and 82.0%(females) for dogs.Twelve metabolites were found in dog plasma, seven were found in the mouse plasma and six were seen in the rat plasma.In all the three species, a tetrahydrofuran-ring-opened carboxylic acid metabolite (M482/1) was the most abundant metabolite, which accounted for 39.3% (males) and 5.0% (females) of plasma radioactivity in mice, 30.4% (males) and 17.7% (females) in rats, and 9.4% (males) and 6.6% (females) in dogs.Other relative abundant metabolites included a destetrahydrofuranyl metabolite M380/1 (0.2 -2.4% in mice, 5.3-5.4% in rat and 1.2 -1.8% in dogs), a tetrahydrofuran ring-opened dihydroxyl metabolite M468/1 (0.4 -3.6% in mice, 3.9-5.3% in rat and 0.7 -1.0% in dogs), an oxidation-dehydrogenation metabolite M464/1 (0.4 -3.8% in mice, 4.9 -5.0 % in rats Metabolite Profiles in Plasma and 0.7 -1.1% in dogs), and a glucuronide metabolite M626/3 (0.1 -6.0% in mice, 0.7 -1.1% in dog; not found in rats).The remaining metabolites were much less abundant and each represented for ≤ 5.9% of plasma radioactivity in all three species.These minor metabolites included M498/1 (a triple oxidation/tetrahydrofuran ring-opened metabolite), M482/2 (a carboxylic acid metabolite isomeric to M482/1), an acid metabolite (M464/2), a cyclic ester metabolite (M464/3), a cyclic ester-carboxylic metabolite (M478/1), deschlorination metabolite (M418/1), as well as four glucuronide conjugates (M556/ -Not detected * % Plasma radioactivity is calculated using AUC 0-24h for mice and AUC 0-8h for rats and dogs, ** Remaining radioactivity was contributed by one or more metabolite, each ≤ 1.1% of plasma radioactivity  Metabolite profiles in feces Unchanged empagliflozin found in feces represented 9.5 -10.5% of the administrated dose in mice, 18.0 -21.3% in rats, and 16.3 -19.5% in dogs (Table 3).Representative radiochromatogram of male rat feces is shown in Figure 2. As with plasma and urine, the number of metabolites in feces was higher in dogs than in mice or rats.M482/1 was the most abundant fecal metabolite in all the three species, and accounted for 19.0 -23.6%, 16.3 -21.4%, and 21.0 -25.5% dose in mice, rats and dogs, respectively.The second most abundant fecal metabolite in all the three species was M464/1, accounting for 5.0 -11.7% dose.The remaining metabolites included M380/1, M468/1, M498/1, M482/2, M478/1, M418/1, M464/2, M464/3 and M626/3, and each was ≤ 5.8% of dose.
The contributions to male rat bile radioactivity and percent of radioactive dose of unchanged empagliflozin and its metabolites are provided in Table 3. Biliary excretion accounted for 33.3% of radioactive dose for male rats.In bile, no empagliflozin was found.The majority of the biliary radioactivity (85.2% of bile radioactivity or 28.4% of radioactive dose) was contributed to by metabolite M482/1.The remaining bile radioactivity was accounted for by several metabolites.These bile metabolites were M464/1 (1.3% of radioactive dose), M556/1 (0.7% of radioactive dose), M498/1 (0.3% of radioactive dose) and M380/1(0.4% of radioactive dose) and M468/1(0.2% of radioactive dose).The remaining metabolites each contributed less than 0.2% of the radioactive dose and were not further identified.
Following oral administration of [ 14 C]-empagliflozin to male pigmented rats at 5 mg/kg, distribution of [ 14 C]-empagliflozinderived radioactivity to tissues was limited.The tissue concentrations of empagliflozin-derived total radioacitivity are provided in the supplementary material.Most radioactivities were found in the contents of the gastrointestinal tract and the major organs of excretion.The maximum mean blood and plasma concentrations were observed at 1 hour postdose.Mean concentrations of radioactivity in blood and plasma declined rapidly to low levels at 24 hours postdose and declined below the limit of quantitation Volume 3 | Issue 3 Unchanged empagliflozin in urine contributed to 2.2 -16.2% of administrated dose in mice, 6.9 -10.0% in rats, and 7.1 -9.6% in dogs (Table 3).Representative radio chromatogram of male rat urine is shown in Figure 2. Similar to plasma, more urinary metabolites were found in dogs than in mice or rats and M482/1 being the most abundant metabolite in all the three species.M482/1 accounted for 15.6 -17.5%, 5.9 -9.8% and 6.8 -11.3% of radioactivity dose in the mouse, rat and dog urine, respectively.All other metabolites were each ≤ 4.7% of dose and all of them were also found in plasma.4).In addition, the negative MS/MS showed the loss of the furan ring, corresponding to the m/z 379 ion.These characteristic fragmentations were useful in structure elucidation for metabolites as discussed below.

Metabolite identification
Metabolite identification was based on HPLC retention time, radiochromatography and mass spectral analysis.High-resolution MS, MS/MS and MS 3 analyses were performed for elemental composition determination and structure elucidation, and the results are summarized in  4).M464/1 was thus a product of hydroxylation followed by dehydrogenation.Negative MS/MS of M464/2 gave empagliflozin characteristic fragments.The presence of m/z 383 fragments suggested the presence of a carboxylic group on the glucose moiety.M464/2 was thus identified to be a carboxylic acid metabolite on the 6-position of the glucose moiety.In contrast to M464/1 and M464/2, MS/MS of M464/3 gave M380/1-type fragments such as m/z 379 and 301.The MS/MS data suggested that the [+O-2H] modification in M464/3 was on the tetrahydrofuran ring, which was consistent with a lactone metabolite.

Discussion
Empagliflozin was well absorbed after oral administration in male and female CD-1 mice, male Wistar rats, and male beagle dogs.The clearance of empagliflozin was low in dog and moderate in rat and mouse.Volume of distribution and half-life were generally moderate in all species.Oral bioavailability was moderate in rat (31%) and high in mouse (90-97%) and dog (89%).The difference in oral bioavailability between rats and mice was likely due to two factors.First, the doses were very different, 250 mg/kg in the mouse compared to 5 mg/kg in the rat.The absorption of drug could be saturated at such a high dose.In addition, any transporter efflux or first pass metabolism is likely saturated at 250 mg/kg.
In CD-1 mice and Wistar Han rats, the half-life of empagliflozin-derived radioactivity after IV administration was <1 h for both sexes.This was similar to the half-life of empagliflozin, suggesting that most of the circulating radioactivity was parent compound.The terminal t 1/2 of empagliflozin-derived radioactivity after oral dosing at in mice and rats was longer than that of empagliflozin, suggesting that some of the metabolites were longer-lived than parent.The terminal half-life in Wistar Han rats after oral dosing at 5 mg/kg was short, at 1.98 h for males and 3.73 h for females.
Distribution of [ 14 C]-empagliflozin -derived radioactivity to tissues was limited.Most radioactivities were found in the contents of the gastrointestinal tract and the major organs of excretion.The maximum mean blood and plasma concentrations were observed at 1 hour postdose.
Following dosing of [ 14 C]-empagliflozin to dogs, the plasma terminal phase t 1/2 values for radioactivity were 22.0 h in males and 31.2 h in females following IV administration and 3.60 h in males and 5.16 h in females following oral administration.The data show no gender differences in the systemic exposure to drug-derived radioactivity.The shorter half-life observed in orally dosed animals than in intravenously dosed dogs is due to the lower limit of quantitation found in the intravenously dosed animals, which enabled a larger portion of the terminal elimination phase to be measured.Following oral administration, absorption of [ 14 C]empagliflozin derived radioactivity was 102 and 92.1% in males and females, respectively.
The elimination of drug-derived radioactivity in dogs following intravenous and oral administration of [ 14 C]-empagliflozin was similar and occurred primarily in feces, representing a mean of 61.4 to 70.9% of dose.Most of the administered dose was eliminated in the first 24 h post-dose, with means of 71.9% (males) and 82.0%(females) for the IV doses and 83.6% (males) and 75.4% of dose (females) for the oral dose.Similarly, fecal elimination was the major route in rats (69.4 -79.7% dose) and mice (79.4 -81.7%), and there were no apparent gender differences.
Metabolite profiles in plasma, urine, bile and feces were similar across mice, rats and dogs, although more metabolites were formed in dogs.The most abundant metabolite in all three species was the oxidation metabolites M482/1.Several minor metabolites were found in dog but not in mouse and rat.For each species, no apparent gender differences in metabolic pattern were observed.
In human healthy subjects, unchanged empagliflozin was the most abundant drug-related component in plasma (79.6% total plasma radioactivity), urine (23.7% dose) and feces (34.2% dose) following a single oral dose of [ 14 C]-empagliflozin [14].No major circulating metabolites, but only several minor metabolites, were detected in the human plasma.These minor metabolites included three glucuronide conjugates (M626/1, M626/2, M626/3), M482/1, M464/1 and M468/1.No single metabolite was therefore considered major in humans that could require non-clinical safety assessments, as defined by its presence in plasma at more than 10% of total drug-related material at steady state, which is specified by the FDA Guidance on "Safety Testing of Drug Metabolites" [15] and the ICH Guidance on "Nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals, M3 (R2)" [16].Compared to humans, metabolic pathways involving oxidation were much more prominent in mice, rats and dogs.In addition within the C 14 -labeled ADME studies conducted for empagliflozin, M380/1, M464/2, M464/3, M478/1, M482/2, and M556/1 were found only in animal species.
Biotransformation of empagliflozin in mice, rats and dogs mainly involved oxidation as well as glucuronide and glucose conjugation (Figure 4).Oxidation on the tetrahydrofuran ring followed by ring-opening, possibly via an aldehyde intermediate (not detected), led to the formation of dihydroxyl metabolite M468/1 and its glucose conjugate M630/1, carboxylic acid metabolites M482/1 and M482/2, and subsequent metabolites M463/3 and M478/1.Oxidation on the tetrahydrofuran ring followed by O-dealkylation resulted in the formation of M380/1, which further gave glucuronide conjugate M556/1.Oxidation on the glucose moiety resulted in the formation of carboxylic acid metabolite M464/2.Oxidation was also responsible for the formation of M464/1.In addition, empagliflozin underwent direct glucuronide conjugation, to a much lesser extent, to form M626/1, M626/2 and M626/3.

Conclusion
Empagliflozin was well absorbed after oral administration in CD-1 mice, Wistar rats, and beagle dogs.The clearance of empagliflozin, determined following IV administration, was low in dogs.The volume of distribution and half-life of total radioactivity were generally moderate in all species.The oral bioavailability of total radioactivity was moderate in rats and high in mice and dogs.Excretion patterns were generally similar in males and females across species evaluated.Feces was the primary route of excretion of [ 14 C]-empagliflozin-derived radioactivity.Distribution of [ 14 C]-empagliflozin-derived radioactivity to tissues was limited.Most radioactivities were found in the contents of the gastrointestinal tract and the major organs of excretion.The maximum mean blood and plasma concentrations were observed at 1 hour postdose.There was no binding to melanain.
Metabolite profiles in plasma, urine, bile and feces were similar across mice, rats and dogs.Biotransformation of empagliflozin mainly involved oxidation as well as glucuronide and glucose conjugation.Several minor metabolites were found in dog but not in mouse and rat.All the human metabolites found in a human ADME study were present in the animal species.

Empagliflozin:
The radioactive peak eluting at approximately 59.0 minutes in all matrices corresponded to empagliflozin, as it had a similar retention time, identical MS and MS/MS patterns as authentic standard.The molecular ion was found at m/z 451 in positive mode and m/z 449 in negative mode, both with an isotope ratio of roughly 0.35 for [M+2]/M which was characteristic for a molecular ion containing one chlorine atom.As shown in Figure3, the positive MS/MS of empagliflozin produced several abundant fragment ions, which included fragments at m/z 415 (-2H 2 O), 397 (-3H 2 O), 385 (2H 2 O-CH 2 O), 379 (-4H 2 O), 367 (-3H 2 O-CH 2 O) and the glucose ring-opened fragments at m/z 373, 355, 331 and 287.The majority of these fragmentations were also observed in the negative MS/MS as indicated by the presence of negative fragments at m/z 413, 395, 383, 371, 359 and 329 (Table

Table 1 :
Percentage of radioactive dose recovery in mice, rats and dogs

Table 2 :
Relative distribution (%AUC) of empagliflozin and metabolites in plasma from mice, rats and dogs after oral dosing of [ 14 C] empagliflozin

Table 3 :
Relative distribution (% dose) of empagliflozin and metabolites in urine, feces and bile from mice, rats and dogs after oral dosing of [ 14 C] empagliflozin

Table 4 .
As the metabolite profiling was conducted over a span of 5 years in different Boeheringer Ingelheim laboratories and different lots of HPLC columns were used, retention time shifted from study to study.The retention times from the last profiling experiments are reported in the present paper.by 72 hours postdose.All tissues with measurable concentrations of radioactivity had observed C max at the first sampling time at 1 hour postdose.The matrices with the highest concentrations of drug-derived radioactivity were gastrointestinal tract contents, urine, and bile.The highest average tissue C max values were measured in liver, renal cortex, kidney, renal medulla, and cecum with values of 4060, 2890, 2540, 2050, and 1470 ng equivalents/g, respectively.Radioactivity concentrations in central nervous system tissues protected by the blood-brain barrier (cerebellum, cerebrum, medulla, olfactory lobe, and spinal cord) were not measurable at any sampling time.[ 14 C]-empagliflozin-derived radioactivity was not associated with the melanin-containing tissues in the eye or skin.Elimination was complete for all tissues by 72 hours postdose.By the final sampling time of 168 hours postdose, no radioactivity was detected in any measured matrix.Terminal t 1/2 values for radioactivity could be estimated in 11 of the 52 matrices analyzed, with values ranging from 2.05 hours in liver to 8.86 hours in renal cortex.

Table 4 :
Mass spectral analysis of empagliflozin metabolites in mice, rats and dogs * Mass spectral data were obtained in positive mode for M464/1 and M418/1 and negative mode for all other metabolites and empagliflozin It corresponded to a negative molecular ion at m/z 497, 48 daltons (+3O) more than empagliflozin.The negative MS/ MS gave fragments at m/z 395, 377, 359, 299, and 257 (table 4), suggesting addition of 32 daltons (2O) to the furan ring and 16 dalton (O) to one of the aromatic rings.Based on the mass spectral analyses, M498/1 was tentatively identified to be an oxidized metabolite of M482/1 or M482/1.

Table 4 )
. The mass spectral data were consistent with the dechlorination structure.