The administration of dipeptidyl peptidase IV (DPP-4) inhibitors to diabetics has been proven to augment endogenous glucagon-like peptide-1 (GLP-1) activity, which in turn produces a clinically significant lowering of diabetic glycemia comparable with that observed when GLP-1 is administered by direct infusion (Gutniak et al., 1992, 1997; Deacon et al., 1995; Mentlein, 1999; Drucker, 2003; Mest and Mentlein, 2005). Vildagliptin (Galvus, LAF237, 1-[[3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine) is a potent, orally active inhibitor of DPP-4, which has been shown to ameliorate hyperglycemia in diabetic patients by preventing the cleavage and inactivation of GLP-1. Vildagliptin is now commercially available in the European market (Villhauer et al., 2003; He et al., 2007).

The IC₅₀ of vildagliptin against the DPP-4 enzyme is 2 nM, based on the in vitro recombinant DPP-4 assay, indicating high potency. In humans, the effect of vildagliptin on DPP-4 inhibition is reflected in an IC₅₀ of 4.5 nM, a value that suggests higher potency than that reported for another DPP-4 inhibitor, sitagliptin (IC₅₀ of 26 nM) (Herman et al., 2005; He et al., 2007). To aid in the selection of appropriate species for preclinical testing, the disposition of vildagliptin was evaluated in the rat and dog and compared with that in the human. Results from in vivo absorption, metabolism, and excretion studies in rat and dogs as well as metabolism data from in vitro liver slices are discussed herein. In addition, the metabolism in DPP-4-deficient rats to elucidate the hydrolysis mechanism is discussed. The absorption, metabolism, and excretion of vildagliptin in humans are reported in the accompanying article (He et al., 2009).

Materials and Methods

Chemicals. [¹⁴C]Vildagliptin was synthesized by the Isotope Laboratory of Novartis Pharmaceuticals Corporation (East Hanover, NJ). For the rat study, the specific activity and purity of the compound were 3.1 to 3.2 μCi/mg and >98%, respectively. For the dog study, the specific activity and purity of the compound were 3.23 μCi/mg and 98.4%, respectively.

SDZ 272-885 and DPP728 were used as internal standards for the analysis of unchanged vildagliptin and were obtained from Novartis Pharmaceuticals Corporation. Synthetic standards M20.2, M20.7, and M15.3 were also obtained from Novartis Pharmaceuticals Corporation. The chemical structures of radiolabeled vildagliptin, its related compounds, and internal standards are shown in Fig. 1.

Animal Studies

All animal studies were conducted with approval of the Novartis Animal Care and Use Committee. For both intravenous and oral administration routes, vildagliptin was dissolved in sterile water, unless otherwise specified. Animals were not fasted before dosing.

HanWistar Rat Studies. Male HanWistar rats (200–250 g), age 6 to 8 weeks were used in the studies. All rats had free access to food and water and were housed individually in properly marked metabolism cages.

In the intravenous or oral disposition study, two groups of six rats were administered 100 mg/kg [¹⁴C]vildagliptin, either orally (n = 3) or by slow intravenous injection (n = 3) into the jugular vein, which was exposed surgically under light isoflurane anesthesia. The first group of six animals was used for absorption and excretion studies (group A), and the second group of six was used for biotransformation and metabolism studies (group B). For rats from group A dosed orally (n = 3) blood samples were collected from each rat in heparinized micropipettes. Blood was obtained from the cut tail of each rat at 0.083 (intravenously dosed rats only), 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, and 96 h postdose. For the animals from group B for biotransformation studies, blood was collected from the cut tail of each rat at 0.083 (intravenously dosed rats only), 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h postdose. The blood collected from the three rats at each time point was then pooled and centrifuged to harvest plasma. For the rats in group A, urine and feces were collected from each rat quantitatively during the 0 to 24, 24 to 48, 48 to 72, and 72 to 96 h postdose intervals. The urine samples were collected over ice up to 72 h. After the final collection, each cage was rinsed with water. All samples were stored frozen at or below –20°C until analysis. The radioactivity of all samples was determined by liquid scintillation counting.

DPP-4-Deficient Fischer 344 Rat Studies. The excretion study of vildagliptin was also carried out in male Fischer 344 DPP-4(+) and DPP-4(–) rats after a single intravenous dose of 3 mg/kg [¹⁴C]vildagliptin. The specific activity and radiochemical purity of the compound was ∼47 μCi/mg and >98%, respectively. Three male Fisher 344 DPP-4(+) and three male 344 DPP-4(–) rats (∼250 g) were used. All rats had free access to food and water. Rats were housed individually in properly marked metabolism cages. Urine and feces were collected quantitatively for the 24-h period before dosing and the 0 to 24, 24 to 48, and 48 to 72 h postdose intervals for each animal individually. At 72 h, after the final collections, the rats were sacrificed by appropriate procedures. All samples were stored frozen at or below –20°C until analysis. The radioactivity of all samples was determined by liquid scintillation counting.

Dog Studies. Male beagle dogs (9–15 kg) were used in this study. The dogs had access to water ad libitum and were fed a standardized canine diet. The oral dose was prepared in hand-filled gelatin capsules. The intravenous dose was prepared as a sterile solution of [¹⁴C]vildagliptin in normal saline for injection. The dogs were dosed intravenously (3 mg/kg; 9.7 μCi/kg; n = 2) or orally (6 mg/kg; 19.4 μCi/kg; n = 3) with [¹⁴C]vildagliptin. Blood samples (4 ml) were collected from each dog at 0 (predose), 0.083 (intravenous dose only), 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 168 h postdose. The total blood volume collected did not exceed 1% of the body weight of animals. Blood samples were collected in heparinized syringes via an indwelling catheter in the cephalic vein for up to 12 h and with a butterfly from the cephalic or saphenous veins at the rest of time points. Plasma was obtained by centrifugation at 3000g. Urine and feces were collected from each of the dogs quantitatively in 24-h intervals up to 168 h postdose. Urine was collected under ice cooling for the first 48 h postdose. All samples were stored frozen at or below –20°C until analysis. The radioactivity of all samples was determined by liquid scintillation counting.

Analytical Methods

Determination of Total Radioactivity. For all animal studies, concentrations of total radioactivity in plasma, urine, and cage wash were determined after addition of 2 to 10 ml of NEN Formula 989, followed by mixing. Liquid scintillation counting was done using a Packard spectrometer (Tri-Carb 2500; Canberra Industries, Meriden, CT). Fecal samples were first homogenized with approximately 3 volumes of water. Aliquots of feces homogenates were then combusted with a biological oxidizer before liquid scintillation counting.

The radioactivity in the dose was defined as 100% of the total radioactivity. The radioactivity at each sampling time for urine and feces was defined as the percentage of dose excreted in the respective matrices. The radioactivity measured in plasma was converted to nanogram-equivalents of vildagliptin based on the specific activity of the dose.

Analysis of Unchanged Vildagliptin. Concentrations of vildagliptin in plasma were determined by LC-MS/MS after protein precipitation (rat) or solid-phase extraction (dog). For the solid-phase extraction method, aliquots of dog plasma (100 μl diluted with 100 μl of water) and 200 μl of internal standard (IS) solution (DPP728) were transferred to individual wells in a 1-ml, 96-well polypropylene plate. Extraction of the samples was performed using a Quadra-96 model 320 workstation (Tomtec, Hamden, CT). Before extraction of samples, a 10-mg Oasis HLB 96-well solid-phase extraction plate (Waters, Milford, MA) was conditioned with 300 μl of methanol, followed by 300 μl of water. The samples were applied to the preconditioned extraction plate. The plate was washed with 300 μl of 5% methanol (containing 2% ammonium hydroxide), 300 μl of 20% methanol (containing 2% ammonium hydroxide), and 300 μl of water. After each well was vacuum-dried, the samples were eluted with two 75-μl washes of 80% methanol (containing 0.1% trifluoroacetic acid) and evaporated under nitrogen (35°C) to a volume of ∼50 μl using an Evaporex solvent evaporator (Apricot Designs, Monrovia, CA). The samples were diluted with 50 μl of 15% methanol (containing 0.5% ammonium hydroxide) and mixed before injection.

Rat plasma (50 μl) was treated with 50 μl of internal standard solution (SDZ 272-885) prepared in acetonitrile containing 0.2% formic acid to precipitate proteins. Samples were vortexed and centrifuged. A 10-μl injection of the supernatant was directly injected onto an LC-MS/MS system for analysis.

Samples were quantitated on either a PE Sciex API3000 (Applied Biosystems, Foster City, CA) or a Micromass Quattro LC (Waters) operated in multiple reaction monitoring mode with electrospray ionization as an interface. For dog studies, vildagliptin and IS were separated on a Polaris C18-A column (4 μm, 50 × 2.0 mm, maintained at 45°C) (Metachem Technologies, Torrance, CA) with isocratic elution. The mobile phase of A:B (1:3, v/v) was used, where A is methanol-10 mM ammonium acetate, pH 8.0 (5:95, v/v), and B is acetonitrile-methanol (10:90, v/v). The flow rate was maintained at 0.2 ml/min with an injection volume of 10 μl. For the rat, vildagliptin and IS were separated on a Keystone BetaBasic C8 50 × 2.0 mm column with gradient elution (0 min at 5% mobile phase B, hold for 0.2 min, to 95% B at 2.2 min, hold 1 min, and back to original condition in 0.5 min; flow rate of 0.2 ml/min). The mobile phase A contained 10 mM ammonium acetate with 0.1% formic acid, and B was acetonitrile.

The reaction monitoring transition for vildagliptin was m/z 304.2 → m/z 154.1. For internal standards SDZ 272-885 and DPP728, the reaction monitoring transitions were 210 → m/z 86.0 and 299.2 → m/z 146.1, respectively.

The peak area ratios of vildagliptin to the internal standard were plotted as a function of the nominal concentrations of vildagliptin. The standard calibration curves were constructed by fitting the weighted (1/x, where x = concentration) data points to a quadratic equation using least-squares. The slope, intercept, and coefficient of determination were generated to assess the performance of the assay. The plasma standard curve range was 5 to 500 ng/ml in rat plasma and 5 to 5000 ng/ml in dog plasma. Spiked quality control samples were prepared in control plasma or urine using the reference standard for vildagliptin. Quality control samples were analyzed with study samples to establish assay accuracy and precision. All bioanalytical runs were judged acceptable on the basis of criteria established before the beginning of sample analysis. Likewise, criteria for selection of samples to be repeated were established before the beginning of sample analysis.

Processing of Samples for Metabolite Investigation. Plasma samples were protein-precipitated with acetonitrile-ethanol (90:10, v/v) containing 0.1% acetic acid, and protein was removed by centrifugation. The supernatant was evaporated to near dryness under a stream of nitrogen using the Zymark TurboVap LV (Zymark Corp., Hopkinton, MA), and the residues were reconstituted in acetonitrile-5 mM ammonium acetate containing 0.1% trifluoroacetic acid (10:90, v/v). Aliquots of concentrated plasma extracts were injected onto the HPLC column. Representative (equal percentage of the volume) pooled urine samples were prepared for analysis. An aliquot was centrifuged and injected onto the HPLC column without further purification. Feces homogenates were pooled at equal percent weight for each subject and extracted twice with methanol by vortexing and centrifugation. Aliquots of combined supernatant (5 ml) were evaporated to dryness under a stream of nitrogen using the Zymark TurboVap LV, and the residues were reconstituted in acetonitrile-5 mM ammonium acetate containing 0.1% trifluoroacetic acid (10:90, v/v). Aliquots of concentrated fecal extracts were injected onto the HPLC column.

HPLC Instrumentation for Metabolite Pattern Analysis. Vildagliptin and its metabolites in urine, plasma, and feces were analyzed by HPLC with online or offline radioactivity detection using a Waters Alliance 2690 or 2795 HPLC system (Waters) equipped with either a Phenomenex Synergy Hydro-RP column (4 μm, 4.6 × 150 mm, maintained at 30°C; Phenomenex, Torrance, CA) and a guard column of the same type or a YMC ODS-AQ C18 column (3 μm, 4.6 × 150 mm, maintained at 35°C) and a guard column of the same type. The mobile phase consisted of 5 mM ammonium acetate containing 0.1% trifluoroacetic acid (pH 2.3) (solvent A) and acetonitrile (solvent B). For analysis performed on the YMC ODS-AQ C18 column (HanWistar rats and Beagle dogs), the mobile phase was initially composed of 100% solvent A and held for 4 min. The mobile phase composition was then linearly programmed to 14 to 19% solvent B over 21 to 25 min and held for 0 to 3 min and to 95% solvent B in 1 min and held for 4 to 5 min. The mobile phase condition was returned to the starting solvent mixture over 1 min. The system was allowed to equilibrate for 10 min before the next injection. A flow rate of 0.9 or 1.0 ml/min was used for analysis. For analysis performed on the Synergy Hydro-RP column [rat Fisher DPP-4(+) male and rat Fisher DPP-4(–) male], the mobile phase was initially composed of 100% A and held for 4 min. The mobile phase composition was then linearly programmed to 13% solvent B over 26 min and held for 2 min, to 60% B in 0.5 min and held for 4 min, and to 95% solvent B in 0.5 min and held for 4 min. The system was allowed to equilibrate for 10 min before the next injection. A flow rate of 1.0 ml/min was used for analysis.

For online radioactivity detection, a flow-through radioactivity detector equipped with a 250-μl liquid cell (β-RAM; IN/US, Tampa, FL) with a scintillant (Flo-Scint II liquid scintillant) flow rate of 4.5 ml/min was used. For offline radioactivity detection, the HPLC effluent was fractionated into a 96-deep well LumaPlate (PerkinElmer Life and Analytical Sciences, Waltham, MA) using a fraction collector (FC 204; Gilson Inc., Middleton, WI) with a collection time of 8.4 s/well. Samples were dried under a stream of nitrogen, sealed, and counted for 1 to 15 min/well on a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences).

The amounts of metabolites of parent drug in plasma or excreta were derived from the radiochromatograms (metabolite patterns) by dividing the radioactivity in the original sample in proportion to the relative peak areas. Parent drug or metabolites were expressed as concentrations (in nanogram-equivalents per milliliter) in plasma or as a percentage of dose in excreta. These values are to be considered as semiquantitative only, in contrast with those determined by the validated quantitative LC-MS/MS assay.

Structural Characterization of Metabolites by LC-MS/MS. LC-MS/MS instruments used for metabolite characterization included the following: 1) A Finnigan LCQ ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) equipped with an electrospray ion source. The effluent from the HPLC column was split, and approximately 500 μl/min was introduced into the atmospheric ionization source after diverting to waste during the first 4 min of each run to protect the mass spectrometer from nonvolatile salts. The electrospray interface was operated at 5000 V, and the mass spectrometer was operated in the positive ion mode. CID studies were performed using helium gas at the collision energy of 35% (arbitrary unit). 2) A two-channel Z-spray (LockSpray) of a time-of-flight mass spectrometer (Q-TOF Ultima Global; Waters, Manchester, UK). Leucine enkephalin was used as the mass reference standard for exact mass measurement and was delivered via the second spray channel at a flow rate of 10 μl/min. The mass spectrometer was operated at a resolution of ∼8000 m/Δm_FWHM with spectra being collected from 120 to 800 amu. The ionization technique used was positive electrospray. The sprayer voltage was kept at 3000 V, and the cone voltage of the ion source was kept at a potential of 50 V. For the time-of-flight MS/MS experiments, collision energies of 24 to 27 eV were used with argon as the collision gas. 3) A PerkinElmer Sciex API3000 triple quadrupole mass spectrometer equipped with a TurboIonSpray probe operating at 375°C. Full scan spectra were typically obtained from 100 to 650 m/z using a 0.2-amu step size. Positive ion spectra were obtained using ion spray voltage of 4500 V and orifice voltage of 40 V with ring voltage of 190 V. A collision energy of 35 eV was used for MS/MS experiments, with a nitrogen collision gas setting of 4.

In vitro blood distribution and protein binding in rat and dog. Triplicate aliquots of the stock solution in ethanol containing [¹⁴C]vildagliptin (35 × 10⁶ dpm) and known amounts of unlabeled vildagliptin were spiked to 1 ml of human fresh blood or plasma (n = 3) to achieve final concentrations of 10 to 10,000 ng/ml.

For the blood distribution study, triplicate determinations of the hematocrit in blood were made and triplicate pooled blood samples (1 ml) were prepared from male HanWistar rats (n > 3) or male Beagle dogs (n = 3). After gentle mixing, a single 200-μl aliquot of blood containing [¹⁴C]vildagliptin was pipetted for radioactivity analysis. Then the blood samples were incubated at 37°C for 30 min and centrifuged at ∼3000g for ∼15 min. at 37°C. The resultant plasma was analyzed for radioactivity using a single 200-μl aliquot.

For the protein binding study, triplicate pooled blood samples (1 ml) were prepared from male HanWistar rats (n > 3) or male Beagle dogs (n = 3). The pH was adjusted to 7.4 by adding 10 μl of 0.1 N HCl/ml plasma and gently vortexing the sample. After a single aliquot of plasma containing [¹⁴C]vildagliptin (200 μl) was pipetted for radioactivity analysis, each sample (∼0.8-ml aliquot) was transferred to the sample reservoir of an individual micropartition centrifuge tube (Centrifree Micropartition Centrifuge Tube; Millipore Corporation, Billerica, MA). The membrane had a molecular mass cutoff of 30,000 Da. The driving force for ultrafiltration was provided by centrifugation using a fixed-angle rotor for 20 min at ∼1000g at 37°C. The ultrafiltrate contained the free fraction, and 200-μl aliquots were analyzed for radioactivity. Nonspecific binding studies were conducted in 0.2 M sodium phosphate buffer (pH 7.4) under the same conditions described above.

For radioactivity analysis of blood/plasma samples in the blood distribution study, 200 μl of blood was pipetted onto individual Combusto pads, air-dried, and combusted in a Packard 308 oxidizer before counting in a liquid scintillant. For radioactivity analysis in the protein binding study, aliquots (200 μl) of plasma samples and filtrates were mixed with 2 ml of a liquid scintillant (NEN Formula 989) in a vial for direct counting. The radioactivity in all samples was determined by liquid scintillation counting in a Packard spectrometer.

In vitro metabolism in rat and dog liver slices. [¹⁴C]Vildagliptin was incubated with liver slice preparations from rat and dog. The incubations were performed at 5 and 20 μM substrate concentrations for 1, 2, 4, 8, 18, and 24 h. The incubates were analyzed by HPLC with online radioactivity detection. Metabolites formed from the incubations were characterized by LC-mass spectrometry. The livers of a male Beagle dog and a male HanWistar rat were excised and placed in cold V7 preservation solution. The individual organs were cored transversely and sliced in ice-cold oxygenated (95% O₂-5% CO₂) V7 preservation media. The viability of the rat and dog slices was confirmed over 24 h by determining the intracellular K⁺ content and by measuring lactate dehydrogenase leakage in 0.1% dimethyl sulfoxide- and vildagliptin-exposed slice incubates. The slices were placed onto roller culture inserts and maintained at 37°C in Dulbecco's modified Eagle's-Ham's F-12 media and supplemented with antibiotic antimycotic solution (Invitrogen, Carlsbad, CA), NuSerum, and Mito+ Serum Extender. After a preincubation period of 90 min, fresh medium containing [¹⁴C]vildagliptin in 0.1% dimethyl sulfoxide was added. At the various time points, the slice and medium were transferred to separate vials, and the roller culture vial and insert were bathed in methanol, which was then collected. Before HPLC analysis, the liver slices were disrupted by homogenization with methanol-H₂O (50:50) followed by brief sonication. The incubation medium was extracted with methanol, and the methanol wash was evaporated to dryness. All fractions were pooled, and the protein was pelleted at approximately 40,000g for 10 min at 20°C. The pellet was reextracted with methanol, and the resultant supernatant was evaporated to dryness and combined with the pooled sample.

Pharmacokinetic Analysis. Pharmacokinetic variables were determined by fitting the concentration-time profiles with a two-compartmental model for intravenous (parent) and noncompartmental model for intravenous (parent) and oral (parent and metabolites) administration. (WinNonlin software version 4.0; Pharsight, Mountain View, CA). With the noncompartmental model the following was determined: area under the plasma drug concentration-time curve between time 0 and time t (AUC_0–t); AUC until time infinity (AUC_0–∞); highest observed plasma drug concentration (C_max); time to highest observed drug concentration (t_max); apparent terminal half-life (t_1/2); and steady-state volume of distribution of parent drug (V_ss) calculated as (Dose) · (AUMC)/(AUC)², where AUMC is the area under the first moment of the concentration-time curve, and clearance (CL) is calculated as Dose/AUC_0–∞. For compartmental models, the percentage of areas under the curve, contributed by the distribution and elimination phases with α and β, were estimated using the following equation: The absorption was estimated on the basis of the ratio of percent dose excreted in the urine after intravenous administration compared with that after oral administration using the following equation:

Results

In Vitro Blood Distribution and Protein Binding in Rats and Dogs. The mean plasma/blood ratio (C_b/C_p) was 1.0 and 1.1 in rats and dogs, respectively, indicating approximately equal distribution between plasma and blood cells. The distribution was independent of concentration between 10 and 10,000 ng/ml.

The mean plasma protein binding of vildagliptin in rats and dogs was low (8.1 and 3.2%, respectively) and also independent of concentration. The nonspecific binding of the compound to centrifuge tubes and/or membranes was low (<12%), suggesting that ultrafiltration is a suitable method.

Pharmacokinetics of Vildagliptin and Total Radioactivity. The pharmacokinetic parameters based on total radioactivity in plasma after a single oral dose in rats and dogs are summarized in Table 1. The intravenous and oral pharmacokinetic parameters of vildagliptin in rats and dogs are summarized in Tables 2 (intravenous) and 3 (oral). The concentration versus time profiles of radioactivity and vildagliptin in plasma after a single intravenous or oral dose in rats and dogs are summarized in Fig. 2. Vildagliptin was rapidly absorbed with a mean t_max between 0.5 and 1.5 h (Table 3). After an oral dose of 100 mg/kg [¹⁴C]vildagliptin to rats, absorption was estimated to be 65 to 69% with a moderate bioavailability (45%). In the dog after a single 6 mg/kg oral dose, the extent of absorption was complete, resulting in a high oral bioavailability (100%). The high absorption and bioavailability are consistent with the absolute bioavailability observed in humans (85%) (He et al., 2009).

After an intravenous dose, the plasma or blood clearance of vildagliptin (C_b/C_p ratio ∼1) was 2.9 l/h/kg in the rat and 1.3 l/h/kg in the dog (Table 2). The volume of distribution at steady-state was 8.6 and 1.6 l/kg in the rat and dog, respectively. The distribution and elimination half-lives were 0.57 h (82% of AUC) and 8.8 h for the rat and 0.05 and 0.9 h (87% of AUC) for the dog.

Excretion of Radioactivity and Unchanged Vildagliptin. The urinary and fecal excretion data after a single dose of radiolabeled vildagliptin in rats and dogs are summarized in Table 4. After a single intravenous or oral dose, urinary excretion after 168 h accounted for 40.7 to 47.6% and 69 to 72.4% in the rat and dog, respectively. After both dosing routes, fecal excretion was 32.2 to 57.3% in rat and 6.74 to 11.3% in dog. After an intravenous dose, unchanged vildagliptin was excreted in both urine and feces of rat (∼41% of the dose) and in urine of dog (∼19% of the dose). The total recovery was 80.9 to 100% for rat and 83.5 to 88.4% for dog.

Metabolite Profiling and Identification. Representative radiochromatograms of rat and dog plasma after oral administration of [¹⁴C]vildagliptin are shown in Fig. 3. Representative radiochromatograms of rat and dog urine and feces after oral administration of vildagliptin are shown in Fig. 4.

Plasma.Table 5 summarizes the AUC percent of vildagliptin and identified metabolites in rat and dog plasma after oral administration of [¹⁴C]vildagliptin. Unchanged vildagliptin was a major circulating component in both animal species after an oral dose, accounting for 22.8 to 31.7% of the AUC. After an oral dose to rats and dogs, the predominant circulating metabolite was M20.7 (a carboxylic acid), accounting for 53.9 and 33.0% of the AUC, respectively. Another major metabolite was M15.3 (a carboxylic acid) in dogs (26.3% of the AUC).

Excreta.Table 6 summarizes the metabolites of vildagliptin detected in rat and dog urine after oral administration of [¹⁴C]vildagliptin. Table 7 summarizes the metabolites of vildagliptin detected in rat and dog feces after oral administration of [¹⁴C]vildagliptin.

Renal excretion of unchanged vildagliptin was one of the major elimination pathways, accounting for 15.5 and 13.6% of the oral dose, in rats and dogs, respectively. In rats, the major metabolic pathway was the hydrolysis at the cyano moiety (M20.7), accounting for ∼26% of the dose in excreta. Minor pathways included glucuronidation (M20.2), monohydroxylation (M14.9), and hydrolysis at the amide bond (M15.3). In dogs, the major metabolites were the hydrolysis products, M20.7 and M15.3, accounting for approximately 26 and 15% of the dose in excreta, respectively.

In the Fischer 344 DPP-4(+) and DPP-4(–) rat study after the intravenous dose of [¹⁴C]vildagliptin, the total amount of unchanged vildagliptin in the excreta (urine and feces) accounted for 30.9% of the dose in the DPP-4 normal substrain and a higher amount (43.5%) in the DPP-4-deficient substrain. In contrast, metabolite M20.7 accounted for 48.0% of the dose in the normal substrain but only 38.4% in the deficient substrain (relatively 20% lower).

Metabolite Characterization by Mass Spectrometry. Metabolite structures were characterized by LC-MS/MS using a combination of full and product ion scanning techniques. The structures of major metabolites, where possible, were supported by comparisons of their retention times on HPLC and mass spectra with those of synthetic standards (vildagliptin, M20.2, M20.7, and M15.3). Vildagliptin displayed a protonated molecular ion (MH⁺) at m/z 304. The product ion spectrum of m/z 304 (Fig. 5) showed major fragment ions at m/z 97, 154, 151, and 133. The ion at m/z 154 corresponded to the amino acetyl pyrrolidine carbonitrile moiety and m/z 97 corresponded to the pyrrolidine carbonitrile moiety. The ion at m/z 151 is from the hydroxy adamantyl ring; neutral loss of water gave an ion at m/z 133. Comparison of metabolite fragment ions with those of vildagliptin allowed assignment of region of biotransformation. The characteristics and proposed structures of major vildagliptin metabolites in preclinical species are summarized in Table 8. The metabolic pathway of vildagliptin in rats, dogs, and humans is shown in Fig. 6.

In Vitro Metabolism in Rat and Dog Liver Slices. Incubation of [¹⁴C]vildagliptin with rat and dog liver slices formed metabolites M20.7, M20.2, and M15.3. The calculated rate of metabolite formation, expressed as nanomoles per hour per milligram wet tissue, for the various metabolic pathways at 5 μM was in the following rank order: rat > dog > human (He et al., 2009).

Discussion

The pharmacokinetics of vildagliptin are consistent in rats and dogs, and there are little apparent species disconnects, which allowed accurate prediction of human pharmacokinetics. After an intravenous dose, the plasma clearance of vildagliptin (C_b/C_p = 1) was relatively high for the rat (2.9 l/h/kg) and dog (1.3 l/h/kg) compared with their hepatic blood flows (Davies and Morris, 1993). To project human clearance, allometric scaling was performed (Mahmood, 1999). The relationship between CL (milliliter per minute) and body weight (BW) (kilograms) can be expressed by CL = 31.6 BW^0.78. For a 70-kg human subject, the human CL for vildagliptin was predicted to be 14 ml/min/kg (0.8 l/h/kg), which was only slightly higher than that observed in human after an intravenous dose (0.6 l/h/kg) (He et al., 2009). The in vivo CL rank order with rat (high) > dog (moderate) > human (moderate) was similar to that observed from in vitro liver slice metabolism data, where the metabolic intrinsic clearance at 5 μM concentration was rat > dog > human, thus yielding an in vitro-in vivo correlation. V_ss was moderate to high compared with total body water, 1.6 and 8.6 l/kg in rats and dogs, respectively, indicating a wide tissue distribution. This finding was confirmed by a tissue distribution study in the rat using the whole-body autoradiography technique (H. He and D. Flood, unpublished data). The distribution and elimination half-lives of vildagliptin were short in dogs, whereas in rats the elimination half-life was relatively long (8.8 h). However, the distribution half-life was short in rat (0.57 h), accounting for 82% of the AUC, suggesting that the distribution half-life should be mainly considered to predict multiple dose pharmacokinetics. These results further indicate that an accumulation of the drug after multiple doses is rather unlikely. Circumstantial evidence is also provided from toxicokinetic studies as well as clinical findings, for which no accumulation of vildagliptin was observed after multiple doses (100 mg/day) for 28 days (He et al., 2007).

In general, vildagliptin was rapidly absorbed with a short t_max between 0.5 and 1.5 h in both animals and humans (He et al., 2009) and a high percentage of drug absorbed, yielding a moderate to high bioavailability in both species (45–100%). Vildagliptin has a high aqueous solubility (>50 mg/ml) and moderate permeability (1.5 × 10^-6 cm/s) observed in an in vitro Caco-2 cell culture study (I. Hanna and N. Alexander, unpublished internal data). Thus, solubility is probably not the rate-limiting step in the absorption process. The true effective permeability in vivo could be higher than that observed in the Caco-2 system, as vildagliptin shows a high aqueous solubility with a relatively low molecular weight (303) and may be partially absorbed through the paracellular route. The high absorption and bioavailability of vildagliptin indicated a relatively low first-pass metabolism in rats and dogs.

After an intravenous dose, urinary excretion was the major elimination pathway in rats and dogs, accounting for 47.6 to 72.4% of the dose (Table 4), similar to the findings in humans (85.4%) (He et al., 2009). After an oral dose, a similar excretion pattern was seen in dogs, whereas in the rat, the drug-related radioactivity was equally excreted in urine and feces. This was probably due to unabsorbed material in feces (lower absorption in rats than in dogs). After both dosing routes, fecal excretion occurred to a lesser extent in dog (6.74–11.3% of the dose). The drug-related radioactivity was excreted rapidly within 24 h for both species. The total recovery was acceptable in rats and dogs (81–100% of the dose).

Vildagliptin was mainly metabolized before excretion in both rats and dogs. One of the major metabolic pathways involved hydrolysis at the cyano moiety to form a carboxylic acid metabolite (M20.7) in rat and dog. The other predominant metabolic pathway included the hydrolysis of the amide bond to form M15.3 (a carboxylic acid) in dog. Renal excretion of unchanged vildagliptin was also a major elimination pathway in rats and dogs. In general, the metabolite profiles were similar between the intravenous and oral dose groups in both animal species. All metabolites observed in humans (He et al., 2009) were also found in the animal species, indicating that the preclinical species studied could be suitable for safety assessment studies.

The parent drug was a major circulating component in rats and dogs, representing 31.7 and 22.8% of AUC after an oral dose, respectively. M20.7 was the predominant circulating metabolite in the rat and dog (33–53.9% of the AUC) and human (55% of the AUC). The second major metabolite was M15.3 in the dog (26.3% of the AUC) and M20.2 (vildagliptin O-glucuronide) in the rat (5.98% of the AUC). In excreta, M20.7 accounted for 26% of the oral dose, respectively, in both rats and dogs, similar to that in humans (57% of the dose). The other major metabolite in excreta was M15.3, accounting for 15% of the oral dose in the dog. The formation of M20.7 or M15.3 does not seem to be mediated by cytochrome P450s, as these metabolites were not formed in liver microsomal incubations (He et al., 2009). In rat, dog, and human liver slices, the carboxylic acid metabolites derived from the hydrolysis pathways (M20.7 and M15.3) and the glucuronic acid conjugate metabolite M20.2 were found, suggesting that the hydrolysis and glucuronidation pathways do occur in the liver. Furthermore, in the Fischer 344 DPP-4(+) and DPP-4(–) rat study after an intravenous dose, the total amount of unchanged vildagliptin in the excreta (urine and feces) accounted for 30.9% of the dose in the DPP-4 normal substrain and a higher amount (43.5%) in the DPP-4(–) substrain. However, metabolite M20.7 accounted for 48.0% of the dose in the normal DPP-4(+) substrain, but only 38.4% of the dose in the deficient DPP-4(–) substrain. These findings suggest that approximately 20% of the cyano group hydrolysis reaction may be attributable to the DPP-4 enzyme. M20.7 was also formed by human recombinant DDP-4 in vitro, supporting the contribution of this enzyme to this metabolic transformation (He et al., 2009). M20.2 was found to be primarily catalyzed by UGT2B7, with minor contributions by UGT2B17 and UGT2B4 (He et al., 2009).

The following metabolites were found in rats or/and dogs but not in humans, including M14.9 (hydroxylation at the adamantyl ring), M16.7 (hydroxylation at the pyrrolidine ring), M17.4 (hydroxylation at the pyrrolidine ring of M20.7), M18.6 (amide metabolite resulting from hydrolysis of the cyano moiety), M14.2 (amide metabolite), and M17.7 (a monohydroxy acid metabolite resulting from ring opening of the pyrrolidine ring). M21.6, found in dogs and humans, could stem from the decarboxylation of M20.7 and/or the loss of cyano group of the parent. However, this pathway was minor in all species (≤2% of the dose).

In conclusion, the pharmacokinetics of vildagliptin in rats and dogs were characterized by rapid oral absorption and elimination, combined with moderate to high bioavailability. Human clearance and pharmacokinetics could be predicted on the basis of allometric scaling from preclinical data. All metabolites observed in human were also detected in rats and dogs. Vildagliptin is eliminated mainly by metabolism to the acid metabolite from the hydrolysis of the cyano group M20.7 (rat and dog) or acid metabolite from the hydrolysis of amide bond M15.3 (dog). The data also suggest that cytochromes P450 have little involvement in the elimination of vildagliptin.