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The Disposition and Metabolism of Naveglitazar, a Peroxisome Proliferator-Activated Receptor - Dual, -Dominant Agonist in Mice, Rats, and Monkeys

Lilly Research Laboratories, Department of Drug Disposition, Eli Lilly and Company, Indianapolis, Indiana

(Received August 11, 2006; accepted September 26, 2006)

	Abstract

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Naveglitazar [LY519818; benzenepropanoic acid, -methoxy-4-[3-(4-phenoxyphenoxy)propoxy], (-S)-] is a nonthiozolidinedione peroxisome proliferator-activated receptor - dual, -dominant agonist that has shown glucose-lowering potential in animal models and in the clinic. Studies have been conducted to characterize the disposition, metabolism, and excretion of naveglitazar in mice, rats, and monkeys after oral and/or i.v. bolus administration. After oral administration of [¹⁴C]naveglitazar, naveglitazar was well absorbed and moderately metabolized in all species evaluated, with total recoveries of radioactivity ranging from 90 to 96%. Naveglitazar was the most abundant peak observed in circulation at C_max, representing 68 to 81% of the total radioactivity in plasma. The most prominent metabolite observed in circulation was the R-enantiomer of naveglitazar, LY591026, which is formed via enzymatic chiral inversion. para-Hydroxy naveglitazar and the sulfate conjugate of para-hydroxy naveglitazar were also observed in circulation in most species, especially in the monkey. The metabolic pathways observed include enzymatic chiral inversion, aromatic hydroxylation, oxidative dehydrogenation, and/or various phase II conjugation pathways. Naveglitazar was highly bound to plasma proteins among the species examined (>99%), and binding was independent of concentration. Biliary excretion was recognized as the most prominent excretion pathway in bile duct-cannulated rats (79 of the 96% recovered), producing an acyl glucuronide conjugate of naveglitazar and a sulfate and glucuronide diconjugate of para-hydroxy naveglitazar, which were shown to be reversible. The primary excretory pathway observed in mice and monkeys was via the feces. In summary, naveglitazar was well absorbed, moderately metabolized, and excreted via the feces in mice, rats, and monkeys.

Naveglitazar [LY519818; benzenepropanoic acid, -methoxy-4-[3-(4-phenoxyphenoxy)propoxy], (-S)-] (Fig. 1), is a peroxisome proliferator-activated receptor (PPAR) - dual, -dominant agonist. PPAR compounds are members of the nuclear receptor superfamily, and have been shown to play a role in lipid and carbohydrate homeostasis (Keller et al., 1993). PPAR-, which is predominately expressed in adipose tissue, regulates the transcription of genes involved in glucose and lipid metabolism (Auwerx, 1999; Kersten et al., 2000). A class of agents known as thiazolidinediones (TZDs), or glitazones, have been shown to modulate PPAR-, resulting in lower plasma glucose, insulin, triglyceride, and fatty acid levels in humans with type 2 diabetes (Olefsky, 2000; Schoonjans and Auwerx, 2000). Naveglitazar, a non-TZD, functions as a potent and efficacious insulin sensitizer in rodents, possessing a novel profile that may result in an improved therapeutic agent for the treatment of type 2 diabetes and associated dyslipidemia (Liu and Reifel-Miller, 2005). In type 2 diabetics, naveglitazar significantly reduced fasting serum glucose, fasting triglycerides, and hemoglobin A1C, and significantly increased HDL cholesterol, displaying an efficacy comparable to or superior to the currently marketed TZD rosiglitazone (Prince et al., 2004). As we continue to understand the functions of different subtypes of PPAR receptors and the complex metabolic disorders associated with type 2 diabetes, dual-PPAR agonist molecules may become a new class of therapeutic agents that may provide more favorable clinical outcomes for type 2 diabetics (Bond and Yates, 2004; Desvergne et al., 2004; Rizvi, 2004). The objective of this work was to characterize the disposition and metabolism of naveglitazar in mice, rats, and monkeys, which will aid in the safety assessment for this molecule as it relates to clinical practice.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Structures of [14C]naveglitazar (S-enantiomer), LY591026 (R-enantiomer), and [13C]internal standard (compound 487748).

	Materials and Methods

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Materials. Naveglitazar (S-enantiomer, LY519818), [¹⁴C]naveglitazar (Fig. 1), LY591026 (R-enantiomer), internal standard (compound 487748), 4-hydroxy-naveglitazar (LY621631), O-sulfate of 4-hydroxy-naveglitazar sodium salt (LY2291229), and dehydrogenated naveglitazar (LY2419535) were synthesized at Eli Lilly and Company (Indianapolis, IN). The radiochemical purity of [¹⁴C]naveglitazar was approximately 98% as determined by HPLC and the specific activity was 13.8 µCi/mg. [³H]Naveglitazar sodium (in a solution of 19272.7 ng/ml) was obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). 4, 4'-Dihyroxydiphenyl ether was obtained from TCI America (Portland, OR). 4-Hydroxydiphenyl ether was obtained from Aldrich Chemical Company (Milwaukee, WI). The ß-glucuronidase (type HP-2, from Helix pomatia) was purchased from Sigma Chemical Co. (St. Louis, MO). Ultima Flo M was purchased from PerkinElmer Life Sciences (Boston, MA). All other reagents and solvents were of analytical grade and were obtained commercially.

Animal Experiments. All animal experiments were conducted according to protocols approved by the Eli Lilly Animal Care and Use Committee. All animals were acclimated to experimental conditions before use (at least 3 days for mice and rats, and at least 6 weeks for monkeys). Food and water were supplied ad libitum throughout the acclimatization and experimental periods. The oral dose suspensions used in the studies were prepared with 1% carboxymethylcellulose sodium, 0.5% sodium lauryl sulfate, 0.085% povidone, and 0.05% Dow Corning Antifoam 1510 US in purified water. The dose solution used for intravenous administration in monkeys was prepared with 40% PEG 300, 60% purified water, and 0.8% 1 N NaOH.

Dosing of Animals and Sample Collections. The single-dose plasma pharmacokinetics of naveglitazar, LY591026, and radioactivity were studied in male ICR mice, male Fischer 344 rats, and cynomolgus monkeys after oral administration of [¹⁴C]naveglitazar. A separate study was conducted in rats to evaluate the pharmacokinetics of naveglitazar and LY591026 after the administration of a single 10-mg/kg dose of each compound. Mice and rats were administered a 10-mg/kg oral dose of [¹⁴C]naveglitazar with a specific activity of 50 µCi/kg. Monkeys were given a 1-mg/kg intravenous bolus (i.v.) or a 5-mg/kg oral dose of [¹⁴C]naveglitazar with a specific activity of 10 µCi/kg.

Mice were divided into three groups. Group 1 animals (n = 3 per time point) were housed in shoebox cages and were used to determine the pharmacokinetics of naveglitazar, LY591026, and radioactivity. Blood samples were collected for plasma from mice via cardiac puncture at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h postdose. Group 2 mice were housed in metabolism cages (n = 3 per cage) for the collection of excreta to determine the mass balance and metabolism of naveglitazar. Urine, feces, and cage wash were collected in 24-h intervals for up to 120 h. The third group of animals (n = 3 per time point) were housed in shoebox cages and used to evaluate the metabolites present in plasma.

Rats were divided into three groups. Group 1 animals (n = 3 per time point) were housed in shoebox cages and were used to determine the pharmacokinetics of naveglitazar, LY591026, and radioactivity. Blood samples were collected for plasma from rats via cardiac puncture at 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h postdose. Group 2 rats (n = 4) were individually housed in metabolism cages for the collection of excreta to determine the mass balance and metabolism of naveglitazar. Urine, feces, and cage wash were collected in 24-h intervals for up to 120 h. Group 3 animals, which were bile-cannulated rats (n = 4), were individually housed in metabolism cages for the collection of excreta to determine the mass balance and metabolism of naveglitazar. Urine, feces, and cage wash were collected in 24-h intervals for up to 72 h, whereas bile was collected in 8-h intervals. In a separate study, rats were given naveglitazar or LY591026. In this study, blood samples for plasma were collected at 0.5, 1, 2, 4, 8, 12, and 24 h to determine pharmacokinetics of each analyte.

Monkeys were divided into two groups. Group 1 animals (n = 4, 3 female and 1 male) received an oral dose and group 2 (n = 4, 3 female and 1 male) received an i.v. dose of [¹⁴C]naveglitazar. All animals were housed in individual metabolism cages for collection of excreta. Blood samples were collected for plasma via the femoral vein at 0.083, 0.17, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 168, 240, 336, 408, 504, 576, and 672 h postdose after i.v. dosing and at 1, 2, 4, 8, 12, 24, 48, 72, 96, 168, 240, 336, 408, 504, 576, and 672 h postdose after oral dosing. Urine, feces, and cage wash were collected in 24-h intervals for up to 672 h postdose. For all studies, blood was collected in tubes containing EDTA as the anticoagulant, and plasma was obtained by centrifugation and stored at approximately –70°C until subsequent analysis.

Radioactivity Analysis. Radioactivity in plasma, urine, bile, and cage wash was determined by mixing aliquots of plasma, urine, bile, or cage wash (0.1 g) with approximately 12 ml of Beckman Ready Protein Plus (Beckman Coulter, Fullerton, CA) or Packard Aquassure scintillation fluid (Canberra Industries, Meriden, CT) and counted in a liquid scintillation counter (Beckman Coulter LS5000TD). Mouse and rat carcasses were gently boiled in a beaker containing ethanol and potassium hydroxide until all tissues were dissolved. Aliquots were mixed with scintillation fluid and acetic acid and then counted in a liquid scintillation counter. Radioactivity in fecal homogenates, which were prepared by soaking fecal samples in a 1:1 methanol/water (v/v) solution that was frozen, thawed, and then shaken vigorously, was measured by scintillation counting of trapped ¹⁴CO₂ after combustion of dried homogenate aliquots. Samples were combusted on a Packard Sample Oxidizer (model 307; PerkinElmer Life and Analytical Sciences).

Plasma Protein Binding. Plasma protein binding of naveglitazar was investigated using [³H]naveglitazar and an ultracentrifugation technique. Mouse, rat, and monkey plasma was spiked at concentrations of 0.1, 1, 10, 100, and 1000 ng of [³H]naveglitazar/ml and incubated for 1 h at 37°C. After incubation, three 1.2-ml aliquots were centrifuged at 130,000 rpm for 3.25 h at 37°C. Aliquots of the unbound fraction were diluted with 10 ml of scintillation fluid and analyzed by liquid scintillation counting.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Recovery of naveglitazar-related material in the excreta after oral administration of [14C]naveglitazar in mice, rats, and monkeys. , mouse; , intact rat; , bile-cannulated rat; , monkey.

Metabolism Sample Preparation. Plasma and excreta samples from the single-dose pharmacokinetic and excretion studies were used to assess the metabolism of naveglitazar. Plasma samples were extracted by protein precipitation with acetonitrile. The extraction was conducted by adding acetonitrile (3 parts) to plasma (1 part, v/v). After the samples were mixed and centrifuged, the supernatants were transferred to silanized glass tubes, evaporated to dryness under nitrogen, and reconstituted with a 50:50 methanol/water solution. Urine samples were either directly injected (for those samples with high amounts of radioactivity) or extracted by solid-phase extraction (SPE) using C18 or Waters Oasis MAX cartridges (1 cc; Waters, Milford, MA). For C18 SPE (ISI Isolute, 100 mg; Jones Chromatography, Lakewood, CO) extraction, the cartridge was conditioned with 1 ml of methanol followed by 1 ml of water. After conditioning, 1 ml of urine was loaded onto the cartridge, followed by a washing step with 1 ml of water. The sample was then eluted with 1 ml of methanol, which was then concentrated to approximately 1/10 of the original volume by evaporation under nitrogen. For Waters Oasis MAX SPE, the cartridge was conditioned with 1 ml of methanol followed by 1 ml of water. The urine sample was acidified with concentrated HCl (20 µl of HCl was added to 1 ml of urine) and the acidified urine (1 ml) was loaded into the preconditioned cartridge. The cartridge was washed with 1 ml of 50 mM ammonium acetate/methanol (95:5). The sample was eluted with 1 ml of 2% formic acid in methanol. The eluted sample was evaporated to dryness under nitrogen and reconstituted in methanol/water (50:50). Bile samples were directly injected after centrifugation. Fecal samples were extracted with methanol. A 1- to 3-g fecal homogenate was added to 2 to 6 ml of methanol, vortexed, mixed on a rotator for approximately 15 min, and then centrifuged. The supernatant was transferred to a silanized glass tube. The fecal extraction procedure was then repeated and the second supernatant was added to the first supernatant. The combined supernatant was then evaporated to dryness under nitrogen and reconstituted in methanol/water (50:50).

Metabolite Radioprofiling by HPLC. Metabolites in various matrices were separated on a reverse phase HPLC column with a Shimadzu LC-10 system (Shimadzu, Kyoto, Japan). Mobile phase A consisted of 90% water, 5% methanol/acetonitrile (50:50), 5% isopropanol alcohol with 10 mM ammonium formate. Mobile phase B consisted of 5% water, 90% methanol/acetonitrile (50:50), 5% isopropanol alcohol with 10 mM ammonium formate. For plasma and fecal extracts from rats and monkeys, and urine from monkeys and mice, a Discovery C18 column (4.6 mm x 150 mm, 5 µm; Supelco, Bellefonte, PA) was used with the following gradient: 0 to 35 min, linear gradient from 0% to 75% B; 35 to 36 min, ramping to 90% B; 36 to 40 min, isocratic at 90% B; 40.0 to 40.1 min, ramping from 90% to 0% B; 40.1 to 50 min, isocratic at 0% B. The flow rate was 1 ml/min. To improve the separation of acyl glucuronide isomers, metabolites in rat bile were separated on a Keystone Fluophase RP column (2.1 mm x 150 mm, 5 µm; Thermo Electron Corporation, Waltham, MA) with the gradient set as 0 to 3 min, isocratic at 10% B; 3 to 40 min, linear gradient to 50% B; 40.0 to 40.1, ramping to 90% B; 40.1 to 50 min, isocratic at 90% B; 50 to 50.1 min, ramping from 90% B to 10% B; 50.1 to 55 min, isocratic at 10% B. For mouse plasma and fecal extracts, a Luna C18 column (4.6 mm x 150 mm, 3 µm; Phenomenex, Torrance, CA) was used to improve the separation of LY519818 and its taurine conjugate. The gradient was programmed as 0 to 35 min, linear gradient from 20% to 75% B; 35 to 45 min, isocratic at 75% B; 45.0 to 45.1 min, ramping to 90% B; 45.1 to 50 min, isocratic at 90% B; 50.0 to 50.1 min, ramping from 90% B to 20% B; 50.1 to 57 min, isocratic at 20% B. The metabolites were detected by either a radiochemical detector (Berthold Z 5004; Berthold Technologies, Bald Wildbad, Germany) equipped with a 500-µl liquid cell (PerkinElmer Ultima Flo M) and scintillation fluid set at a flow rate of 3.5 ml/min or by HPLC fraction collection with subsequent radioactivity counting and chromatogram reconstruction. For fraction collection, HPLC effluent was collected into PerkinElmer 96-well LumaPlates at 15-s intervals. The plates were dried under centrifugal vacuum and counted on a Packard Microplate Scintillation and Luminescence counter (TopCount NXT; PerkinElmer Life and Analytical Sciences). The radiochromatograms were reconstructed using the radioactivity (cpm or dpm) versus time data.

LC/MS and LC/MS/MS. LC/MS/MS analyses were conducted on an ion trap mass spectrometer (Finnigan LCQ Deca; Thermo Electron Corporation) equipped with a Shimadzu LC-10 HPLC system. The mobile phases and gradients used in LC/MS analysis for all matrices were the same as those used for radioprofiling. Smaller-diameter HPLC columns (2.1 mm x 150 mm) and slower flow rates (0.25 ml/min) were applied to improve the sensitivity of LC/MS analysis. Mass spectral analysis was performed with both positive and negative ion electrospray ionization. The capillary temperature was 225°C and the spray voltage was 5 kV. The full-scan MS spectra were obtained from m/z 200 to m/z 1000. The MS/MS spectra were obtained with the collision energy set at 45%.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Plasma concentration versus time profiles of naveglitazar, LY591026, and naveglitazar-derived radioactivity in mice, rats, and monkeys after administration of [14C]naveglitazar. , total 14C; , naveglitazar; , LY591026.

NMR Sample Preparation and Analysis. Naveglitazar and the M7 metabolite (dehydrogenated parent) were separately dissolved in 150 µl of deuterochloroform and transferred to 3-mm NMR tubes. Data were acquired on a Varian INOVA 500 NMR spectrometer (Varian, Inc., Palo Alto, CA) using a Varian gradient, triple-resonance IFC probe (LY519818) or a Varian gradient, triple-resonance, Cold Probe. The suite of experiments included a standard proton, homonuclear gradient correlation spectroscopy, direct-correlation multiplicity-edited heteronuclear single quantum collisionally activated dissociation, and long-range gradient heteronuclear multiple-bond collisionally activated dissociation. Data were referenced to the solvent at 7.27 ppm for ¹H and 77.2 ppm for ¹³C. The air-dried sample of M7 was later dissolved in 150 µl of deuteromethanol for the acquisition of a gradient Bird Carr Purcell Meilboom Gill-heteronuclear single quantum multiple bond correlation for the measurement of long-range heteronuclear coupling constants.

The M1 metabolite (sulfate conjugate of the para-hydroxyl metabolite) was isolated from the rat liver slices incubated with naveglitazar for 24 h. Column trapping was used to introduce the metabolite into the flow probe of the NMR apparatus. The metabolite was isolated and transferred to a 2-ml HPLC injector loop connected to the LC/NMR apparatus. The LC apparatus was programmed to pump deuterium oxide at 2 ml/min. Using a flow splitter, 25% of this flow went through the loop and was then combined with the remainder of the flow in a 50-µl static mixer. From the mixer, the flow was directed to a secondary trapping column (Thomson Instrument Company, Clear Brook, VA; Thomson Liquid Chromatography, 30 x 3 mm, TIC-PAQC 18, 5 µm), where the metabolite was retained. The transfer of the analyte to the trapping column was monitored with the UV detector. When the transfer was complete (i.e., the UV absorbance returned to baseline), the flow was stopped, the mixer was removed, and the trapping column was reoriented such that the flow through the column was reversed. The mobile phase was subsequently changed to 50% deuterium oxide and 50% deuterated acetonitrile to elute the metabolite from the secondary column and into the flow probe of the NMR apparatus. NMR data for M1 were acquired on a Varian INOVA 600 NMR spectrometer equipped with a triple-resonance flow probe. The suite of experiments included a standard proton with water elimination through transverse gradients, homonuclear wet double quantum filtered correlated spectroscopy and total correlation spectroscopy, and through-space rotating frame nuclear Overhauser enhancement spectroscopy. Data were referenced to deuterated acetonitrile at 1.94 ppm for ¹H and 1.39 ppm for ¹³C.

Chiral Assay for LY621631 (M2). M2 (para-hydroxy naveglitazar) was isolated from mouse, rat, and monkey fecal extracts using a C18 HPLC column. The S- and R-enantiomers of this metabolite were separated via chiral chromatographic techniques using a chiral HPLC column (Thermo Electron Chiral/ß-OH, 1 mm x 250 mm, 5 µm). Mobile phase A consisted of 73:25: 2:0.05 acetonitrile/methanol/water/5 M ammonium formate, and mobile phase B consisted of 60:38:2:0.05 acetonitrile/methanol/water/5 M ammonium formate. The gradient was programmed as follows: 0 to 0.5 min, isocratic at 0% B; 0.50 to 10 min, linear gradient from 0% to 20% B; 10.0 to 10.1 min, ramping to 100% B; 10.1 to 15.0 min, isocratic at 100% B; 15.0 to 15.1 min, ramping from 100% to 0% B. The run time was 25 min. The flow rate was 250 µl/min. Peak detection was carried out by UV using a Surveyor PDA detector and by full-scan MS and MS/MS using an ion trap mass spectrometer (Finnigan LCQ Duo; Thermo Electron Corporation) in positive electrospray ionization mode with a spray voltage of 5 kV, capillary temperature of 225°C, and collision energy of 45%.

Analytical Procedure. Naveglitazar and LY591026 in plasma were assayed using a validated 96-well SPE procedure with LC/MS/MS detection. Specifically, 10 µl of internal standard (2500 ng of compound 487748/ml) was added to sodium EDTA plasma (75, 100, or 200 µl for mouse, rat, or monkey, respectively), followed by the addition of 700 µl of 5:95 methanol/1% formic acid solution (v/v). A dry molecular sieve was added to each well of the collection block. The following steps on the procedure were performed on a Tomtec Quadra 96-well workstation (model 320; Tomtec, Hamden, CT). Each well of the ANSYS C18 15-mg disk extraction block was conditioned with 250 µl of methanol followed by 300 µl of 5:95 methanol/formic acid (v/v). Each mixed sample was transferred to the SPE block and the samples were aspirated through the cartridge at an approximate flow rate of 200 µl/min. The cartridge bed was washed with 400 µl of water followed by 250 µl of 23:75:0.1 methanol/water/5 M ammonium formate (v/v). Full vacuum was applied for 30 s. A polypropylene collection block containing molecular sieves was placed into the vacuum manifold, and the analytes were eluted from the cartridges with 150 µl of acetonitrile at an approximate flow rate of 250 µl/min. The collection block was removed from the Tomtec Quadra workstation. The analyst then added 250 µl of acetonitrile that was previously dried with molecular sieves to the collection block. A cover film was applied to the collection block and the samples were allowed to stand for a minimum of 2 h. The collection block was then centrifuged at approximately 3000 rpm for 5 min. Extracts were transferred to a new block and then centrifuged at approximately 3000 rpm for 5 min. Extracts were placed in autosampler vials for analysis using selected reaction monitoring turbo ion spray LC/MS/MS in the negative ion mode (naveglitazar and LY591026 m/z = 421.1 389.2, LY487748 m/z = 427.1 395.1). The LC/MS/MS instrument used was a Sciex API-3000 (Applied Biosystems/MDS Sciex, Foster City, CA). The validation range of the assay for naveglitazar and LY591026 was approximately 1 to 5000 ng/ml in rat and monkey plasma and 2 to 5000 ng/ml in mouse plasma. The mean accuracy (relative error) for naveglitazar and LY591026 for all species evaluated was within 17% of the theoretical values across the entire standard curve range. The mean precision (%CV) for all species was 12%. Plasma samples exceeding the upper limit of quantitation were determined by dilution with control plasma.

View larger version (20K): [in this window] [in a new window]   FIG. 4. HPLC-UV chromatograms of liver slice homogenates from rat, monkey, and human after in vitro incubations with naveglitazar.

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Percentage of protein binding of naveglitazar was calculated as follows: % Protein Binding = (1–C_f/C_p) x 100, where C_f = amount of radioactivity in protein-free fraction of plasma sample and C_p = amount of radioactivity in plasma sample.

	Results

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Excretion of Radioactivity in Animals. Excreta from mice, rats, and monkeys were analyzed for radioactivity to assess the mass balance. After oral administration, the mean recovery was high (Table 1; Fig. 2). Fecal elimination was the primary excretory route observed in all species evaluated. Furthermore, bile duct-cannulated rats excreted the majority of the dose via the bile with low levels (7%) of the dose in the feces. Low levels of radioactivity were observed in the urine of rodents (up to 3.5%), whereas higher levels were recovered in monkey urine (18% of the dose). The excretory pathway was similar in monkeys given an i.v. bolus dose of [¹⁴C]naveglitazar (data not shown).

Pharmacokinetics of Naveglitazar, LY591026, and Radioactivity in Animals. The pharmacokinetic data for naveglitazar (S-enantiomer), LY591026 (R-enantiomer), and radioactivity are shown in Table 2. The plasma concentration versus time profiles are illustrated in Fig. 3. [¹⁴C]Naveglitazar was quickly absorbed and moderately metabolized before elimination. After oral administration, 47, 31, and 62% of the radioactivity, as assessed by AUC values, were circulating as metabolites in mice, rats, and monkeys, respectively. LY591026 represented 8, 13, and 5% of the circulating metabolites in mice, rats, and monkeys, respectively, or 3 to 4% of the total AUC. Half-lives for naveglitazar and radioactivity were similar within each species; however, monkeys had substantially longer half-lives in comparison to mice and rats. Naveglitazar and radioactivity were slowly cleared from the system circulation in all species evaluated. The clearance of LY591026 appeared to be faster than that of the parent compound; however, the calculated half-lives were likely underestimated because of the lack of quantifiable plasma concentrations of LY591026 in the terminal elimination phase.

In a separate study, rats received either a 10-mg/kg dose of naveglitazar (S-enantiomer) or LY591026 (R-enantiomer). The resulting pharmacokinetic parameters, which are shown in Table 3, indicate that both analytes are observed in the systemic circulation after the administration of either molecule.

Plasma Protein Binding. The extent of in vitro binding of [³H]naveglitazar to plasma proteins was evaluated by ultracentrifugation in mouse, rat, and monkey plasma. The mean percentages ± S.E.M. of protein binding of radioactivity in plasma over the concentration range of 0.1 to 1000 ng/ml after in vitro incubation at 37°C for 60 min were 99.5% ± 0.1% (mice), 99.6% ± 0.1% (rat), and 99.6% ± 0.3% (monkey). These results show that naveglitazar is highly bound to plasma proteins among the species examined, and binding is independent of concentration.

In Vitro Metabolic Profiles from Liver Homogenates. The major metabolic pathway observed in human liver slices incubated in the presence of naveglitazar was the para-hydroxy metabolite (M2) with subsequent sulfate conjugation to M1 (Fig. 4). In addition, dehydrogenation of the 2-methoxy propanoic acid chain was observed as a minor metabolic pathway, which led to the formation of M7. Small amounts of a sulfate conjugate of dehydrogenated naveglitazar + oxygen (M9) were also observed. The in vitro metabolic pathways observed in rat and monkey liver slices were similar to that in human liver slices. All metabolites observed in human liver slices were found in rat and monkey liver slices.

Metabolic Profiles of Excreta from Animals. Unchanged naveglitazar, the para-hydroxy metabolite (M2), and a dehydrogenated metabolite (M7) were observed in fecal extracts from all species evaluated (Table 4; Fig. 5). M2 was the most prominent metabolite observed in fecal samples from mice, intact rats, and monkeys. M2 was isolated from fecal extracts and profiled using chiral chromatography to evaluate the presence of both enantiomers. Both enantiomers were observed across all species with the R-enantiomer, which is formed via chiral inversion, accounting for up to 40% of the total metabolite. These data suggest that when appropriate, both enantiomers of a metabolite may exist.

View larger version (22K): [in this window] [in a new window]   FIG. 5. HPLC radiochromatograms of feces from mice, rats, and monkeys and bile from rats after oral administration of [14C]naveglitazar.

In bile-cannulated rats, small amounts of M2 were observed in feces; however, large amounts of the sulfate conjugate of the para-hydroxy metabolite (M1) were observed in bile (Table 4; Fig. 5). In addition, glucuronide conjugates of naveglitazar (M13a-c), sulfate, and glucuronide diconjugates of the para-hydroxy metabolite (M12a-c), and glucuronide conjugates of a dehydrogenated metabolite (M14a-b) were observed in rat bile. In mouse feces, naveglitazar, M1, M2, M7, dehydrogenated naveglitazar + oxygen (M8), a sulfate conjugate of dehydrogenated naveglitazar + oxygen (M9), and a taurine conjugate (M18) were observed. M1 and M2 were the most abundant metabolites observed.

No naveglitazar was observed in monkey urine (Fig. 6). Small amounts of M1, M2, a glucuronide conjugate of para-hydroxy naveglitazar (M3), and sulfate and glucuronide conjugates of hydroxylated diphenyl ether molecules (M15, M16, M17) were observed in monkey urine. None of these metabolites represented more than 3.5% of the recovered dose (Table 4).

View larger version (11K): [in this window] [in a new window]   FIG. 6. HPLC radiochromatogram of monkey urine after oral administration of [14C]naveglitazar.

Metabolic Profiles of Plasma in Animals. A total of six metabolites were observed in plasma across all species evaluated (Table 5). Naveglitazar represented the most abundant peak observed in circulation in all species (Fig. 7). Small amounts of M1 and a phenyl acetic acid metabolite (M6) were also observed in all species. M2 was observed in monkey and mouse plasma.

View larger version (18K): [in this window] [in a new window]   FIG. 7. HPLC radiochromatogram of plasma from mice, rats, and monkeys after oral administration of [14C]naveglitazar.

Mass Spectrum of Naveglitazar and Major Metabolites. Full-scan MS spectrum of parent drug (Fig. 8) showed the [M + NH₄]⁺ ion at m/z 440. The product ion spectrum of m/z 440 gave the fragment ions at m/z 391 (loss of ammonia and methanol), m/z 377 (loss of ammonia and formic acid), and m/z 345 (loss of ammonia, methanol, and formic acid). Loss of ammonia and 4-phenoxyphenol with subsequent loss of methanol or formic acid produced fragment ions at m/z 237, m/z 205, and m/z 191, respectively. Comparison of the HPLC retention time and product ion mass spectrum with that obtained from the synthetic standard of naveglitazar confirmed the identification of parent peak.

View larger version (17K): [in this window] [in a new window]   FIG. 8. Mass spectra for naveglitazar and major metabolites. Naveglitazar; M2, para-hydroxy naveglitazar (LY621631); M1, sulfate conjugate of para-hydroxy naveglitazar; M12, sulfate and glucuronide diconjugate of para-hydroxy naveglitazar; M6, phenyl acid metabolite; M7, dehydrogenated naveglitazar; M18, taurine conjugate of naveglitazar.

The NMR data for M1 also exhibited correlations very similar to those of naveglitazar. The absence of the H1 triplet and subsequent change of H2 to a doublet is consistent with the substitution of the O-sulfate group at position 1 (Table 6).

M2 (para-hydroxy naveglitazar). Full-scan MS spectrum of M2 gave a [M + NH₄]⁺ ion at m/z 456, corresponding to addition of an oxygen to parent drug. In the product ion mass spectrum of m/z 456, loss of ammonia and methanol with subsequent loss of formic acid produced the fragment ions at m/z 407 and m/z 361. The product ion at m/z 237 corresponds to loss of 4-phenoxyphenol + O, suggesting that hydroxylation occurs on either of the aromatic rings of the di-phenyl ether moiety. Subsequent loss of methanol or formic acid from ion m/z 237 gave product ions at m/z 205 and m/z 191. Comparison of the HPLC retention time and characteristic product ions of M2 with that from a synthetic standard of para-hydroxy naveglitazar (LY621631) confirmed the identification of M2.

M6 (phenyl acetic acid metabolite). Full-scan MS spectrum of M6 gave a [M–H]^– ion at m/z 377. The product ion mass spectrum of m/z 377 produced the fragment ions at m/z 333 (loss of CO₂), m/z 185 (phenoxy phenol anion ion), m/z 151 (4-hydroxy phenyl acetic acid anion), and m/z 107 (loss of CO₂ from the 4-hydroxy phenyl acetic acid anion). Accurate mass measurement for M6 supports the proposed structure.

M7 (dehydrogenated naveglitazar). In positive ion mode, full-scan MS spectrum of M7 gave a [M + NH₄]⁺ ion at m/z 438, corresponding to parent –H2. The product ion mass spectrum of m/z 438 gave fragments at m/z 421 (loss of ammonia) and m/z 403 (loss of ammonia and H₂O). In negative ion mode, full-scan MS spectrum of M7 gave a[M–H]^– ion at m/z 419. The product ion mass spectrum of m/z 419 produced the fragment ions at m/z 375 (loss of CO₂), m/z 343 (loss of CO₂ and methanol), and m/z 185 (corresponding to the 4-phenoxyphenolated anion). The loss of 4-propoxyphenyl phenyl ether with subsequent loss of CO₂ gave the product ion at m/z 149. The existence of ion m/z 149 supports the assignment of the position of the dehydrogenation. The dehydrogenated naveglitazar standards (trans- and cis-) were synthesized. Comparison of the HPLC retention time and product ion mass spectrum indicates that M7 has a cis conformation.

The NMR data for M7 exhibited correlations very similar to those of naveglitazar (Table 6). The aliphatic H19 and H20 resonances were not observed as in naveglitazar. H19 was instead observed as a singlet at 7.08 ppm, indicative of the olefin bond. The long-range heteronuclear ³J_H19-C21 coupling was observed to be 4.5 Hz, indicative of a cis relationship between H19 and the acid moiety (Breitmaier and Voelter, 1990).

M12a-c (sulfate and glucuronide diconjugate of M2). M12a-c was observed in bile from bile duct-cannulated rats dosed with LY519818. Full-scan MS of M12a-c gave a [M + NH₄]⁺ ion at m/z 712 and a [M–H]^– ion at m/z 693 in positive ion mode and negative ion mode, respectively, corresponding to addition of an oxygen, a sulfate, and a glucuronic acid to parent drug. The product ion spectrum of m/z 712 gave a product ion at m/z 632 as a base peak (loss of sulfate). In the product ion spectrum of m/z 693, loss of sulfate or loss of glucuronide or loss of sulfate and glucuronide gave product ions at m/z 613 (base peak), m/z 517, and m/z 437, respectively. The product ion spectra for M12a to M12c were similar, suggesting that they are isomers resulting from the acyl migration within glucuronic acid. After hydrolysis of rat bile with ß-glucuronidase, all three peaks corresponding to M12a-c disappeared, and the peak of 4-hydroxy naveglitazar increased.

M13a-c (acyl glucuronide conjugate of naveglitazar). M13a-c in positive ion mode showed the [M + NH₄]⁺ ion at m/z at 616 (corresponding to additions of an ammonia and a glucuronic acid to naveglitazar) and in negative ion mode showed the [M–H]^– ion at m/z 597 (corresponding to addition of a glucuronic acid to naveglitazar). MS² of m/z 597 gave the aglycone ion at m/z 421 (base peak) and glucuronic acid anion at m/z 175. The MS² spectra for M13a-c were similar, suggesting that they are isomers resulting from the acyl migration within glucuronic acid.

M18 (taurine conjugate of naveglitazar). Full-scan MS spectrum of M18 gave a [M + NH₄]⁺ ion at m/z 547, indicating that the nitrogen content in M18 differs from that in the parent molecule. The product ion mass spectrum of m/z 547 produced the fragment ion at m/z 498 (loss of ammonia and methanol). Subsequent loss of NH₂CH₂CH₂SO₃H (125 atomic mass units) (characteristic fragmentation for a taurine conjugate) from the ion m/z 498 gave the fragment ion at m/z 373. Loss of 4-phenoxyphenol from the ion m/z 498 gave the fragment ion at m/z 312.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Naveglitazar is a novel and potent PPAR - dual, -dominant agonist that may be effective for the treatment of type 2 diabetes. These studies were conducted to characterize the disposition and metabolism of naveglitazar in mice, rats, and monkeys, which will aid in the safety assessment for this molecule as it relates to clinical practice. In addition, in vitro liver slice incubations with naveglitazar were conducted to compare the metabolic pathways between animals and human. The metabolic pathways observed in rat and monkey liver slices were similar to that in human liver slices. Thus, all metabolites observed in human liver slices were found in rat and monkey liver slices.

After administration of [¹⁴C]naveglitazar, drug-related material was well absorbed and slowly eliminated. Naveglitazar was the most abundant peak observed in circulation representing 76, 81, and 68% of the total radioactivity at T_max in the pooled plasma samples from mice, rats, and monkeys, respectively (Table 5). Since the metabolic profiling was conducted using reverse phase chromatographic techniques, these values represent the summation of naveglitazar and LY591026, which is formed in vivo from naveglitazar via chiral inversion. The mechanism for the chiral inversion is not clear; however, it appears to be derived via an enzymatic process. Using rat liver homogenate, LY591026 could only be produced in the presence of the cofactors CoA and ATP. These data are similar to those reported for the enzymatic chiral inversion of ibuprofen, which was mediated by the formation of a thioester CoA intermediate followed by racemization (Knihinicki et al., 1989). In general, LY591026, which has 10-fold less binding affinity for the PPAR- receptor compared with naveglitazar, represents 3 to 4% of the total radioactive AUC in plasma, and 5 to 13% of the circulating metabolites observed across the animal species evaluated (Table 2). The metabolic chiral inversion is not unidirectional. In rats orally administered LY591026, exposure to both naveglitazar (S-enantiomer) and LY591026 (R-enantiomer) was evident.

LY621631 (M2), a phenyl acetic acid metabolite (M6), and/or the sulfate conjugate of M2 (M1) were detected in plasma from all three species. A few other small metabolites were also detected (Table 5). In general, LY591026 was the most abundant metabolite observed in plasma.

The primary route of excretion was the feces. In bile duct-cannulated rats, the majority of naveglitazar-related material was recovered in bile with only 7% of the dose recovered in the feces, indicating very good absorption of the material. The percentage of recovered dose was similar across all species, ranging from 90 to 96% (Table 1), although the rate of excretion varied. In rodents, most of the naveglitazar-related material was recovered within a few days after dosing, whereas the rate of excretion in monkeys appeared to be slower (Fig. 2). It is important to note that 4 to 14% of the dose was recovered in the carcass of rodents after the live-phase portion of the studies. The recovery in monkeys was slower than that in rodents during the first few days, and a long, noticeable elimination phase was observed after the first week of the recovery period. In general, approximately 10% of the dose was recovered during this period of time, which is similar to the percentage of dose recovered in rodent carcasses.

Since fecal elimination represented the major excretory pathway, it is important to evaluate the metabolic pathways observed in feces across the three species evaluated. Naveglitazar represented only 2 to 9% of the total dose administered; thus, the majority of the radioactivity in feces was metabolites. Three major pathways were observed. In bile duct-cannulated rats, phase I oxidation and phase II conjugation resulted in three major metabolites, a sulfate and glucuronide diconjugate of para-hydroxy naveglitazar (M12a-c), a sulfate conjugate of para-hydroxy naveglitazar (M1), and an acyl glucuronide conjugate of naveglitazar (M13a-c). M1 and M12a-c likely result from the same pathway, which accounted for 52.8% of the dose administered. Due to the functional groups located on separate ends of naveglitazar, it is not unexpected to observe a sulfate and glucuronide diconjugate. After the consumption of red wine in humans, the abundant flavonoid catechin is metabolized to some degree to a sulfate and glucuronide diconjugate (Donovan et al., 1999). In addition, resorcinol is partially excreted in rats as a sulfate and glucuronide diconjugate (Kim and Matthews, 1987). M13a-c accounted for 14.2% of the dose. The para-hydroxy metabolite was not observed in bile; however, this metabolite was observed in the feces of intact rats (48.9% of the dose) and monkeys (41.2% of the dose), in which no phase II conjugation was detected. Thus, the conjugate forms were probably deconjugated once they entered the lower intestinal track via the bile duct. This pathway was also observed in the mouse, which represented 32.4% of the dose. An additional direct taurine conjugation of naveglitazar was observed in the mouse. This metabolite was only observed in feces and represented 11.1% of the dose. Six metabolites, none of which were higher than 3.5% of the dose, and no parent compound were detected in monkey urine (Fig. 6).

In summary, the disposition and metabolism of LY519818 was characterized in mice, rats, and monkeys and the in vitro metabolic profile was assessed using liver slices from rat, monkey, and human. In general, the in vitro metabolic profile in human liver slices was similar to rat and monkey, although monkey displayed a slightly wider array of metabolites. In addition, the metabolic pathways observed in vitro were also observed in vivo. After oral administration, naveglitazar was well absorbed and slowly eliminated. Studies in bile duct-cannulated rats indicated that naveglitazar and associated metabolites were primarily eliminated via biliary excretion. The excretion profile was similar across all species evaluated; thus, biliary excretion likely played a large role in mice and monkeys as well. Naveglitazar was metabolized by enzymatic chiral inversion, aromatic hydroxylation, oxidative dehydrogenation, and/or various phase II conjugation pathways (Fig. 9). Although naveglitazar was moderately metabolized through both phase I and phase II metabolic processes, the primary circulating drug-related material was parent compound.

View larger version (18K): [in this window] [in a new window]   FIG. 9. Proposed major metabolic pathway for naveglitazar in mouse (M), rat (R), and monkey (P).

	Acknowledgments

 
We express our appreciation to the animal studies personnel supporting Drug Disposition at Eli Lilly and Company for conducting the animal studies.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.012328.

ABBREVIATIONS: LY519818, naveglitazar [benzenepropanoic acid, -methoxy-4-[3-(4-phenoxyphenoxy)propoxy], (-S)-]; LY591026, R-enantiomer of naveglitazar; LY621631, 4-hydroxy-naveglitazar; LY2291229, O-sulfate of 4-hydroxy-naveglitazar sodium salt; LY2419535, dehydrogenated naveglitazar; HPLC, high performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; LC/MS/MS, liquid chromatography with tandem mass spectrometric detection; AUC, area under the plasma concentration versus time curve; SPE, solid-phase extraction; C_max, maximum plasma concentration; T_max, time to reach maximum plasma concentration; k_e, elimination rate constant; t_1/2, half-life; V_ss, volume of distribution at steady state; V_ss/F, apparent volume of distribution at steady state.

Address correspondence to: Jason T. Johnson, Lilly Research Laboratories, Department of Drug Disposition, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285. E-mail: johnson_jason_t{at}lilly.com

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