Abstract
The pharmacokinetics and metabolism of the l-threo isoleucine thiazolidide dipeptidyl peptidase IV inhibitor, di-[2S,3S]-2-amino-3-methyl-pentanoic-1,3-thiazolidine fumarate (ILT-threo) and its allo stereoisomer (ILT-allo) were evaluated in rats, dogs, and monkeys. Both compounds were well absorbed (>80%) in all species, and most of the dose (>60%) was recovered in urine. Metabolites identified in all species included a sulfoxide (M1), a sulfone (M2), and a carbamoyl glucuronide (M3). For both compounds, parent drug had moderate systemic clearance in rats and dogs (∼20–35 ml/min/kg in both species) and lower clearance in monkeys (∼6–9 ml/min/kg). In rats, M1 was present in systemic circulation in concentrations similar to that of parent drug, whereas in dogs and monkeys, exposures to M1 were higher than for parent drug. In dogs, exposures to the sulfoxide metabolite were ∼2 to 3 times higher after administration of ILT-allo than after administration of ILT-threo. Carbamoyl glucuronidation was an important biotransformation pathway in dogs. Circulating levels of M3 were significant in the dog, and present only in trace levels in rats and monkeys. M3 could be produced in in vitro systems in a NaHCO3 buffer under a CO2-saturated atmosphere and in the presence of UDP-glucuronic acid and alamethicin.
A prolongation of the duration of action of the incretin glucagon-like peptide 1 (GLP-11) can be achieved by inhibiting proline-specific serine protease dipeptidyl peptidase IV (DP-IV) with amino acid analogs. The ubiquitous exopeptidase DP-IV is proline-specific (Mentlein, 1999; Pospisilik et al., 2001) and solely responsible for the hydrolysis of GLP-1 to its inactive product (Schmidt et al., 1986; Suzuki et al., 1989; Pauly et al., 1996; Hansen et al., 1999; Deacon et al., 2000). GLP-1 is involved in regulation of glucose homeostasis, playing an important role in nutrient-stimulated insulin release. In addition, GLP-1 is involved in inhibition of gastric motility and secretion (Pederson and Brown, 1972; Schirra et al., 1996), stimulation of insulin gene transcription and biosynthesis (Drucker et al., 1987; Fehmann and Habener, 1992), promotion of β-cell glucose competence (Huypens et al., 2000), preservation and/or restoration of β-cell function (Zawalich et al., 1993), and stimulation of β-cell differentiation and growth (Hui et al., 2001).
In humans, continuous infusion of GLP-1 or GLP-1 analogs to diabetic patients resulted in normalization of both postprandial and fasting glucose (Rachman et al., 1997). Subchronic (6-week) continuous infusion of GLP-1 resulted in profound and significant decreases in fasting plasma glucose and HbA1c (Zander et al., 2002). In obese Zucker rats, acute treatment with the DP-IV inhibitor NVP-DPP728 provided evidence for increase in plasma-active GLP-1 (Balkan et al., 1999).
Recently, a chronic study conducted in Vancouver diabetic rats with the l-threo isoleucine thiazolidide DP-IV inhibitor, di-[2S,3S]2-amino-3-methyl-pentanoic-1,3-thiazolidine fumarate (ILT-threo), was reported (Pospisilik et al., 2002). These results provided the first demonstration that, in the rat, long-term DP-IV inhibitor treatment causes improvements in glucose tolerance, insulin sensitivity, and β-cell glucose responsiveness. In humans, this compound has been shown to improve postprandial glucose tolerance in both acute and subchronic applications (Hoffman et al., 2002). Stabilization of GLP-1, therefore, has been proposed as a new therapeutic approach for type 2 diabetes.
ILT-threo is a competitive, reversible inhibitor of DP-IV with a Ki of ∼300 nM. The compound is equipotent for both membrane-bound and circulating forms of the enzyme and appears to be selective over other proteases. Its allo stereoisomer (ILT-allo) is also a reversible competitive inhibitor of DP-IV, with potency and selectivity properties that are indistinguishable from those of the threo counterpart; therefore, it may also be an attractive development candidate.
Pharmacokinetics and metabolism studies were conducted with these compounds in rats, dogs, and monkeys to determine whether there are any differences in metabolism or advantages in the pharmacokinetic characteristics between these two stereoisomers that would favor the selection of one stereoisomer over the other one as a development candidate.
Materials and Methods
Test Materials.l-threo Isoleucine thiazolidide [(2S,3S)-3-methyl-1-oxo-1(1,3-thiazolidin-3-yl)pentan-2-amine fumarate (2:1)] (ILT-threo) was purchased from Heumann Pharma (Nuremberg, Germany), its allo stereoisomer (ILT-allo) and valyl thiazolidine fumarate, the internal standard (IS) used in the bioanalytical method, were provided by the Process Research department of Merck Research Laboratories (MRL; Rahway, NJ). The corresponding sulfoxides were supplied by Medicinal Chemistry at MRL. 10-[(N,N-Dimethylamino)-alkyl]-2-(trifluoromethyl)phenothiazine (5-DPT) was provided by Dr. John Cashman (Human BioMolecular Research Institute, San Diego, CA). [Pentyl-1-14C] l-threo and allo isoleucine thiazolidide hemifumarate salts were synthesized by the Labeled Compound Synthesis Group at MRL. The radiochemical purity was >99%. All other reagents were obtained from commercial sources.
In Vivo Metabolism.Metabolism in rats. Nine male Sprague-Dawley rats were administered a single oral dose of [14C]ILT-threo at 20 mg/kg. A group of three rats was euthanized at 2, 6, and 24 h postdose and plasma, liver, and kidneys were harvested. In addition, urine and feces were collected for the 24 h group. In a second study, two groups of three rats each received a single administration of [14C]ILT-threo, either i.v. or p.o., at 10 mg/kg. Plasma was collected predose and at 2, 6, 24, 48, and 72 h postdose. Urine and feces were collected predose, and at the following intervals: 0 to 24, 24 to 48, and 48 to 72 h postdose. For dosing, [14C]ILT-threo and [14C]ILT-allo were dissolved in saline at a concentration of 10 mg/ml, with a specific activity of ∼70 μCi/ml.
Bile duct-cannulated dogs and monkeys. The animal phase of this study was conducted at Charles River Laboratories Discovery & Development Services (Wilmington, MA). Briefly, four male dogs (9–11 kg) and four male monkeys (4–6 kg) were dosed intravenously (1 mg/kg, n = or orally (5 mg/kg, n = 2) with [14C]ILT-threo. A separate group of animals was dosed with [14C]ILTallo. Specimens were collected at specified times (blood) or time intervals (bile, urine, and feces), as follows. Bile: predose, 0 to 4, 4 to 8, 8 to 24, 24 to 48, and 48 to 72 h postdose. Blood for plasma: predose, 1, 2, 4, 8, 24, 48, and 72 h postdose. Urine and feces: predose, 0 to 24, 24 to 48, and 48 to 72 h postdose. For dosing, [14C]ILT-threo and [14C]ILT-allo were dissolved in saline at a concentration of 2 mg/ml. Each dog and monkey received approximately 200 and 150 μCi, respectively.
In Vitro Metabolism.Solutions and reagents for in vitro incubations. A cofactor solution was prepared by mixing 4:2:1 (v/v) stock solutions of 50 mM glucose 6-phosphate (0.705 g in 50 ml of water), 10 mM NADP (0.383 g in 50 ml of water), and 14 U/ml glucose-6-phosphate dehydrogenase. The incubation buffer was prepared by mixing 20:2:12 (v/v) stock solutions of 1 M KH2PO4 (pH 7.4), 0.1 M EDTA (pH 7.4), and 0.1 M MgCl2 with 8 ml of microsomal or S9 protein, diluted accordingly to yield a final concentration of 2 and 10 mg of S9 protein/ml or 2 mg of microsomal protein/ml.
Microsomal and S9 incubations. Human, rat, dog, and monkey liver microsomes and liver, lung, kidney, and jejunum S9 fractions were prepared following procedures described in the literature (Raucy and Lasker, 1991). For microsomal incubations, [14C]ILT-threo and [14C]ILT-allo were incubated in duplicate at 5 and 50 μM with and without NADPH (control) for varying times between 0 and 120 min. For all concentrations and time points, the following treatments were evaluated in all possible combinations: 1) incubations conducted under atmospheric air or under a CO2-rich atmosphere; 2) incubations conducted in the presence of 50 mM sodium bicarbonate buffer (pH 7.5 or 8.8) or potassium phosphate buffer (pH 7.4); 3) incubations conducted in the presence and absence of UDP-glucuronic acid (UDPGA) (2 mM), MgCl2 (2 mM), and saccharolactone (5 mM); 4) incubations in the presence of varying concentrations of detergents [glycerol/lubrol, 0–8%, 1:1 (v/v)] or pore-forming agents (alamethicin, 0–250 μg/mg protein). Reactions were initiated with the addition of cofactor solution after preincubation for 2 min at 37°C. Each incubation (total volume of 200 μl) contained 70 μl of cofactor solution, 128 μl of incubation buffer, and 2 μl of substrate. Reactions were terminated with 2 volumes of 1% formic acid in acetonitrile, cooled over wet ice, and centrifuged. Aliquots (200 μl) of each supernatant were transferred to HPLC vials for analysis.
S9 incubations were carried out at 0, 60, and 120 min with 2 or 10 mg of protein/ml, and with 0, 5, and 50 μM concentration of parent drug, with and without NADPH (control). Each incubation vial contained 350 μl of cofactor solution, 640 μl of incubation buffer, and 10 μl of substrate for a total volume of 1000 μl. After preincubation for 2 min at 37°C, reactions were initiated with the addition of cofactor solution. Reactions were terminated with 2 volumes of 50:50 (v/v) ethanol/acetonitrile, iced, and centrifuged. Aliquots (200 μl) of each incubation vial were transferred to HPLC vials for analysis.
Hepatocyte incubations. Cryopreserved human, dog, and monkey hepatocytes were obtained from In Vitro Technologies Inc. (Baltimore, MD). Rat hepatocytes were isolated using a two-step procedure (Pang et al., 1997). Suspensions contained approximately 1 × 106 viable cells/ml. Duplicate substrate concentrations of 0, 5, or 50 μM were incubated for 0, 1, and 2 h. Reactions were terminated by addition of 2 volumes of 1% formic acid in acetonitrile, iced, and centrifuged. The supernatants were subjected to HPLC analysis as described above.
Recombinant P450 incubations. Recombinant human cytochrome P450 (P450) enzymes were obtained from baculovirus-infected insect cells and were prepared by Dr. Tom Rushmore, Department of Drug Metabolism, Merck Research Laboratories. Recombinant human FMO1, FMO2, and FMO3 were purchased from BD Gentest (Woburn, MA). FMO2 was provided by Dr. John Cashman, Human BioMolecular Research Institute (San Diego, CA). Duplicate [14C]ILT-threo and [14C]ILT-allo concentrations of 0, 1, and 10 μM were incubated with and without NADPH (controls) for 1 h in 50 mM potassium phosphate buffer (pH 7.4). Recombinant human FMOs also were incubated in 50 mM sodium bicarbonate buffer (pH 8.4 and 9.5). Reactions were initiated by the addition of cofactor solution and substrate for P450s and FMOs, respectively, after preincubation for 2 min at 37°C. Each incubation vial contained 40 pmol of enzyme, 70 μl of cofactor solution, 128 μl of incubation buffer (described above), and 2 μl of substrate in a total volume of 200 μl. Reactions were terminated by addition of 2 volumes of 1% formic acid in acetonitrile, iced, and centrifuged. The supernatants were subject to HPLC analysis as described above.
Inhibition experiments. To determine the contribution to metabolism by P450 enzymes, FMOs were inactivated by heat treatment by heating human liver microsomes at 55°C for 60 s and cooling down immediately. After adding 2 U of catalase, microsomes were added to the incubation mixture. To distinguish the contribution to metabolism by FMOs, P450 activity was abrogated by conducting human liver microsomal incubations with NADPH (0.5 mM) in glycine/pyrophosphate buffer, pH 8.5, supplemented with Lubrol PX (0.1%, w/v) and glycerol (10%, v/v). Testosterone and 5-DPT were used as positive controls for P450 and FMO activity. The effect of chemical inhibitors selective for individual P450s on the metabolism of [14C]ILT-threo and [14C]ILT-allo was investigated following the procedures described by Baldwin et al. (1999). The inhibitors sulfaphenazole, quinidine, 4-methylpyrazole, quercetin, and furafylline (1, 5, and 20 μM), and ketoconazole (0.5, 2.5, and 10 μM) were added to incubates containing 1 μM [14C]ILT-threo and [14C]ILT-allo to investigate the involvement of CYP2C9, 2D6, 2E1, 2C8, 1A2, and 3A4, respectively. Metabolism in human liver microsomes in the presence and absence of chemical inhibitors was compared to determine inhibitory activity.
Extraction of Radioactivity for HPLC Analysis. Rat, dog, and monkey feces were homogenized with 3 volumes of water and frozen at -20°C until analysis. Bile, urine, and plasma samples were processed with 5 volumes of acetonitrile, vortexed (10 s), and centrifuged (15 min, 1,850g). The supernatant was transferred to a clean test tube, evaporated under nitrogen, and reconstituted in 1 ml of 10 mM ammonium acetate/acetonitrile (9:1). Total radioactivity levels were determined in neat bile, urine, and plasma, and in reconstituted extracts; recoveries were >90%.
Determination of Total Radioactivity. Total radioactivity was determined by combusting triplicate aliquots of the specimen, followed by determination of radioactivity by direct count in a liquid scintillation analyzer. Total radioactivity in rat, dog, and monkey plasma and urine was determined by direct count.
Liquid Chromatography-Tandem Mass Spectrometry Analysis. Metabolite identification was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The system consisted of a PerkinElmerSciex (Boston, MA) API 3000 triple quadrupole mass spectrometer interfaced to a PerkinElmer HPLC system (PerkinElmer Instruments, Norwalk, CT) equipped with two Series 200 micro pumps and a Series 200 autosampler. The radioactivity was detected on-line using a Flow Scintillation Analyzer (PerkinElmer Life Sciences, Boston, MA) equipped with a 0.5-ml liquid cell. Analyte separation was achieved using a Phenomenex (Torrance, CA) Synergi Polar-RP column (3.0 × 150 mm, 4.0 μm). Mobile phases were A, 10 mM ammonium acetate in water, and B, acetonitrile. The flow rate was 0.5 ml/min. Chromatography conditions were isocratic (8% B) from 0 to 4 min. A linear gradient was applied from 4 to 19 min to 55% B, and from 19 to 24 min to 90% B; conditions remained isocratic (90% B) for 5 min. The LC effluent was split with one-fifth directed to the mass spectrometer and the remaining directed to a Flow Scintillation Analyzer (PerkinElmer Life Sciences). The scintillant (Ultima Flo-M; PerkinElmer Life Sciences) was flowed at 1.2 ml/min (three times the LC flow) for radioactivity detection. LC-MS and LC-MS/MS experiments were conducted using a turbo-ionspray interface scanning in the positive ion mode. Turbo-ionspray interface was operated at a source temperature of 350°C and voltage of 5,000 V. A declustering potential of 28 V, a focusing potential of 350 V, and an entrance potential of -10 V was used. Nitrogen was used for all of the nebulizer, auxiliary, and curtain gases. Postcolumn recovery of radioactivity was obtained on representative samples and determined to be >90%.
Pharmacokinetic Studies.Animal phase. Male Sprague-Dawley rats (∼350–450 g), beagle dogs (∼12–15 kg), and rhesus macaques (∼8–12 kg) were dosed with ILT-threo or ILT-allo intravenously at 1, 5, and 10 mg/kg, or orally at 2, 10, and 20 mg/kg. All studies were conducted using three animals per dose level, except for some of the monkey studies, in which two animals were used. For i.v. and oral dosing, parent drug was dissolved in saline. Heparinized blood was collected at 0, 0.083, 0.25, 0.50, 1, 2, 4, 6, 8, 12, and 24 h. Plasma was obtained by centrifugation and stored at -80°C until analysis.
Bioanalytical method. Plasma concentrations of parent drug and the corresponding sulfoxide metabolite (M1) were determined by LC-MS/MS using a PerkinElmerSciex API 3000 mass spectrometer operated in the positive ion atmospheric pressure chemical ionization mode with multiple-reaction monitoring. The HPLC system interfaced to the mass spectrometer consisted of two PerkinElmer Series 200 micro pumps and a PerkinElmer Series 200 autosampler.
Plasma samples (0.1 ml) were fortified with IS (20 ng) and extracted with 10 volumes of 1% formic acid in acetonitrile. Samples were vortexed and centrifuged at room temperature for 15 min at 1850g. Supernatants were concentrated to dryness, and reconstituted with 0.15 ml of 10 mM ammonium acetate in 95% water/5% acetonitrile, adjusted to pH 5.4 with formic acid, for LC-MS/MS analysis. The reconstituted extracts were chromatographed on a Phenomenex Aqua C18 column (30 × 2 mm, 5 μm) and eluted at 1.2 ml/min using the following gradient: 0% B for 0.2 min, followed by a linear increase to 22.5% B in 1.8 min and to 100% B in 0.2 min. The column was then washed at 100% B for 0.6 min. Mobile phases A and B were 10 mM ammonium acetate containing 5% acetonitrile (pH 4.8) and acetonitrile containing 5% 10 mM ammonium acetate, respectively. The HPLC flow was split and only one-fifth was directed to the mass spectrometer. Under these conditions, M1, parent drug, and IS eluted at 0.96 min, 1.28 min, and 1.72 min, respectively. Quantitation was achieved by monitoring the following multiple-reaction monitoring transitions: m/z 219 → 86 for M1, m/z 203 → 86 for parent drug, and m/z 189 → 72 for IS. Transitions were optimized at a collision energy of 21 eV (RO2-Q0, laboratory frame) and at a collision gas (N2) thickness setting of 4. The heated nebulizer source was operated at 400°C with nebulizer and curtain gas (N2) set to 6 and 10, respectively. The orifice and ring voltages were maintained at 28 and 105 V, respectively. Calibration standards were prepared by adding parent drug and the corresponding sulfoxide metabolite (usually triplicates of 8–12 concentrations ranging from 0.1 to 2,500 ng/ml) and IS (20 ng) to plasma. The lower limit of quantitation for parent drug and M1 was 2 ng/ml.
Synthetic standards were not available for the carbamoyl glucuronide metabolites. Approximate quantification of these metabolites was determined by comparing the peak area of the m/z 203 → 86 transition from the carbamoyl glucuronide, which eluted at 1.45 min, to the signal obtained from the same transition for parent compound. It was previously determined that the carbamoyl glucuronide (m/z 423) is susceptible to in-source fragmentation and was completely fragmented in the atmospheric pressure chemical ionization source even at relatively low temperatures (300°C) (Liu and Pereira, 2002). The relative proportions of carbamoyl glucuronide to parent obtained by this LC-MS/MS approximation versus those obtained by radioactivity determinations in a few selected samples were comparable.
Pharmacokinetic calculations. Pharmacokinetic parameters were calculated by established noncompartmental methods. The area under the plasma concentration versus time curve from 0 to 24 h (AUC0–24) was determined using Watson with linear trapezoidal interpolation in the ascending slope and logarithmic trapezoidal interpolation in the descending slope. The portion of the AUC from the last measurable concentration to infinity (AUC0-inf) was estimated by Ct/kel, where Ct represents the last measurable concentration and kel is the elimination rate constant. The latter was determined from the concentration versus time curve at the terminal phase by linear regression of the semilogarithmic plot. AUCs were normalized to 1 mg/kg (nAUC).
Results
Absorption and Elimination. After p.o. administration of ILT-threo to rats and monkeys, ∼90 and 80% of the administered radioactivity was absorbed and recovered in urine, respectively (Table 1). In dogs, ∼70% of the dose was absorbed, with ∼60 and 10% recovered in urine and bile, respectively. Similar dose recoveries were observed in dogs and monkeys for ILT-allo ([14C]ILT-allo was not administered to rats). For both compounds, most of the radioactivity was recovered within 24 h postdose in all species. No major differences in elimination routes were observed between the i.v. and p.o. doses.
In Vivo Metabolism. The structures of the metabolites observed in rats, dogs, and monkeys were elucidated by turbo-ionspray LC-MS using a combination of full scan (Q1), product ion scan (MS/MS), and D2O exchange techniques as described by Liu et al. (2001). Full scan of the parent compound gave an abundant molecular ion at m/z 203. The collision-induced dissociation (MS/MS) spectrum of m/z 203 showed a predominant fragment ion at m/z 86, which was assigned to the isoleucine moiety (Fig. 1). Thus, the presence of a fragment ion at m/z 86 was used to characterize metabolites with an unmodified isoleucine. The fragmentation patterns of M1, M2, and M3 are described in Fig. 1. ILT-threo was metabolized to a sulfoxide (M1), a sulfone (M2), and a carbamoyl glucuronide (M3). ILT-allo produced the same metabolites with different stereochemistry (Fig. 1). Representative HPLC-radiochromatograms are shown in Fig. 2.
After i.v. and p.o. administration of [14C]ILT-threo to dogs, parent drug, M1, and M3 were the major metabolites observed in plasma, comprising 11 to 17, 43 to 47, and 40 to 42% of the total radioactivity, respectively (Table 2). Similar results were obtained after administration of [14C]ILT-allo, where parent drug, M1, and M3 represented 4 to 13, 45 to 49, and 45 to 46%, respectively, of the total radioactivity in plasma. Formation of the sulfoxide metabolite, M1, was an important metabolic route for both compounds in dogs and was recovered mostly in urine. It represented 43 to 52 and 50 to 55% of the dose administered for ILT-threo and ILT-allo, respectively. Dose recovery of parent drug in urine represented 3 to 5 and 4 to 10% for ILT-threo and ILT-allo, respectively.
Parent drug and M1 were the major drug-related components observed in rat plasma, comprising 65 and 35% of the total radioactivity, respectively, for ILT-threo (Table 2). Formation of the sulfoxide metabolite, M1, was an important metabolic route for ILT-threo in rats; 57 to 64% of the dose was recovered in urine. Approximately 30 to 33% of the dose was recovered as intact parent drug in urine. Carbamoyl glucuronidation was not a major route of metabolism in rats (<4% of the dose administered). Radiolabeled ILT-allo was not administered to rats.
In monkeys, parent drug and M1 represented 51 to 62 and 38 to 48% of the total plasma radioactivity, respectively, following administration of ILT-threo, and 55 to 57 and 40 to 42%, respectively, following administration of ILT-allo (Table 2). Formation of the sulfoxide metabolite, M1, was an important metabolic route for both compounds and was recovered mostly in urine (27–50 and 46–51% of the dose for ILT-threo and ILT-allo, respectively). Approximately 25 to 54 and 25 to 32% of the dose was recovered as intact parent drug in urine for ILT-threo and ILT-allo, respectively. Carbamoyl glucuronidation was not a major route of metabolism in monkeys (<4% of the dose administered).
Formation of the sulfoxide metabolite, M1, was an important metabolic route for ILT-threo in all rats, dogs, and monkeys and for ILT-allo in dogs and monkeys. Dose recovery of parent drug in urine also was a major route of compound elimination for ILT-threo in rats and monkeys, and for ILT-allo in monkeys. In dogs, however, elimination of intact parent in urine was not a major clearance route.
Pharmacokinetics. The i.v. pharmacokinetic parameters after a single dose administration of ILT-threo and ILT-allo to rats, monkeys, or dogs at 1, 5, or 10 mg/kg are displayed in Table 3. The systemic clearance (Clp) was moderate in rats and similar between compounds, and tended to decrease with increasing dose levels (from 27 to 17 and from 28 to 23 ml/min/kg, following administration of ILT-threo and ILT-allo, respectively). A moderate Clp also was observed in dogs; it was similar between compounds and tended to decrease with increasing dose levels (from 33 to 25 and 29 to 21 ml/min/kg, after administration of ILT-threo and ILT-allo, respectively). The systemic clearance was much lower in monkeys, ∼8 to 9 and 6 ml/min/kg across dose levels following administration of ILT-threo and ILT-allo, respectively.
In rats and dogs, AUC0-inf values were slightly more than doseproportional (1–10 mg/kg) for both compounds; this was probably a consequence of the slower Clp of these compounds at the higher dose levels. In monkeys, however, AUC0-inf values were fairly proportional to the dose levels. Within each species, there were no differences in plasma AUC0-inf between compounds.
The Vdss was moderate in all species and similar for both compounds, (∼3–5, 2–3, and 1 l/kg in rats, dogs, and monkeys, respectively). The terminal half-life was fairly short, and similar between species and compounds (∼1–3 h).
The oral pharmacokinetics were evaluated in rats, dogs, and monkeys, at 2, 10, or 20 mg/kg dose levels. For both compounds, oral absorption was rapid, with maximum concentrations in plasma achieved between 0.25 and 1 h postdose at all dose levels in all species (data not shown). AUC0–24h values were more than dose proportional between 2 and 20 mg/kg (Fig. 3), and bioavailability at 2 mg/kg was estimated at >90, 80, and 60% in rat, dog, and monkey, respectively. For both compounds, monkey was the species with the highest systemic exposure to parent drug, which was ∼3 times higher than exposures in dogs and rats.
Estimates of urinary clearance for parent compound and the sulfoxide metabolite (Clu,p and Clu,s, respectively) were obtained from the amount of parent drug or sulfoxide metabolite recovered in urine following i.v. administration, over the exposure to parent drug or sulfoxide metabolite during that interval. For ILT-threo, Clu,p estimates were ∼30 and 10 ml/min/kg for rat and monkey, indicating that most of the Clp was accounted for by Clu,p. These values are larger than the glomerular filtration rate (GFR, ∼2 and 5 ml/min/kg in rat and monkey, respectively), suggesting active secretion of ILT-threo in urine. Similar conclusions were derived for ILT-allo in monkeys (Clu,p for ILT-allo was not obtained in rats). This was not the case in the dogs, for which Clu,p (∼2 ml/min/kg) was less than one-tenth of Clp and lower than GFR (∼6 ml/min/kg) for ILT-threo. For ILT-allo Clu,p was ∼10 ml/min/kg and accounted for approximately one-third of Clp. In dogs, Clu,s was approximately 2 times greater for ILT-threo than for ILT-allo in dogs.
Limited pharmacokinetic parameters were obtained for the corresponding sulfoxide metabolites following p.o. administration of either ILT-threo or ILT-allo at 2, 10, and 20 mg/kg in rats, dogs, and monkeys (Fig. 3). Administration of either ILT-threo or ILT-allo to rats resulted in similar systemic exposures to the corresponding sulfoxide metabolite. Moreover, levels of the sulfoxide metabolites were similar to those of the corresponding parent drug. In rats, exposures to the sulfoxide metabolite were similar following administration of ILT-threo or ILT-allo.
In dogs and monkeys, exposures to the sulfoxide metabolite were higher than the exposures observed for the corresponding parent drug. The highest metabolite-to-parent exposure ratio was observed in the dogs: ∼3 to 5 and 8 to 10 following administration of ILT-threo and ILT-allo, respectively. These ratios were ∼1 and 2 following administration of ILT-threo and ILT-allo in monkeys. In addition, exposures to the sulfoxide metabolite following administration of ILT-allo were ∼2 to 3 times higher than those observed for the sulfoxide metabolite following administration of ILT-threo, resulting in a higher exposure to total circulating drug when ILT-allo was administered.
In dogs, circulating levels of the carbamoyl glucuronide metabolite, M3, were significant, with systemic exposures similar to those observed for parent ILT-threo, and ∼50 to 75% of those observed for parent ILT-allo (Fig. 4). In rats and monkeys, only trace levels of M3 were detected in systemic circulation.
In Vitro Metabolism. When ILT-threo and ILT-allo were incubated in NADPH-fortified rat, dog, monkey, or human liver microsomes, the sulfoxide metabolite was the only major product of the incubations, with a small percentage of the sulfone (M2) also present. M1 was also formed in incubations of ILT-threo and ILT-allo in rat, dog, monkey, or human liver S9 and hepatocytes; the carbamoyl glucuronide metabolite (M3) was not detected (data not shown).
Minimal metabolism was observed in S9 fractions obtained from rat, dog, or monkey lung and jejunum, and the only product detected was the sulfoxide metabolite. In kidney S9, up to 15% of parent drug was converted to sulfoxide, the only product detected, indicating that kidney could be a potential site for extrahepatic metabolism in vivo (data not shown).
Incubations with recombinant human P450s or FMOs indicated that all enzymes tested generated M1 as the major metabolite (Table 4); M2 was a minor component in all incubations. In incubations with recombinant human FMOs, metabolism increased with increasing pH. Heat or chemical treatment to abrogate FMO and P450 activity failed to completely inhibit metabolism, indicating multiple enzyme involvement in the formation of M1 (data not shown).
When rat or dog liver microsomal incubations were performed in 50 mM NaHCO3 buffer under a CO2-saturated atmosphere and in the presence of UDPGA and alamethicin, production of the carbamoyl glucuronide metabolite was observed (Table 5). Carbamoyl glucuronide was not detected in human liver microsomes and only trace amounts were detected in monkey liver microsomes. In addition to CO2, the presence of NaHCO3 in the incubation medium was needed to increase the yield of carbamoyl glucuronide. No significant differences were observed in the production of carbamoyl glucuronide when the pH of the incubation medium was 7.5 or 8.5. Formation of carbamoyl glucuronide in incubations containing alamethicin was ∼2 to 3 times higher than in incubations containing glycerol/lubrol (data not shown). If rat or dog liver microsomes were first incubated in the presence of an NADPH-regenerating system and UDPGA, under atmospheric air, and after 1 h, the atmosphere was saturated with CO2, it was possible to generate approximately equal amounts of the sulfoxide and carbamoyl glucuronide metabolites.
Discussion
The pharmacokinetics and metabolism of ILT-threo and ILT-allo, isoleucine thiazolidide stereoisomers that are equipotent competitive, reversible inhibitors of the serine protease DP-IV, were evaluated in preclinical species. Both stereoisomers were rapidly and well absorbed following oral administration to rats (threo only), dogs, and monkeys and were mostly cleared via renal excretion. Estimates of urinary clearance for parent compound indicated that ILT-threo was actively secreted into urine in rats and monkeys. ILT-allo was also actively secreted in monkeys (Clu,p of ILT-allo was not evaluated in rats). Our data suggested that clearance mechanisms for these two compounds were different in dogs: active transporters are probably not involved in the elimination of parent drug, and hepatic clearance is an important component of systemic clearance. These data also suggested differences in clearance mechanisms between the threo and allo stereoisomers in the dog, with elimination of intact parent compound via urine being a more important route for ILT-allo. In the dog, systemic exposures to the ILT-allo sulfoxide metabolite were 2-fold higher than systemic exposures to the ILT-threo metabolite. Differences in exposures to sulfoxide metabolites following p.o. administration of the allo and threo-stereoisomers were not as pronounced in rats and monkeys. Although the sulfoxide metabolite is devoid of biological activity, its significant amounts in systemic circulation could potentially be associated with off-target liabilities. Levels of the carbamoyl glucuronide were significant and similar to levels of parent drug for ILT-threo, and a fraction of parent drug (∼50–75%) for ILT-allo in dogs. The species differences in the presence of carbamoyl glucuronide in systemic circulation is worth noticing, inasmuch as the carbamoyl glucuronide metabolite was not a significant component of systemic circulation in rats and monkeys.
In summary, no differences in the pharmacokinetics of ILT-threo or ILT-allo were detected in monkeys and rats, with both compounds and their corresponding sulfoxide metabolite mostly cleared via urine and probably via an active transport mechanism. In dogs, however, pharmacokinetic differences were observed between compounds, with Clu,p accounting for a small fraction of Clp for ILT-threo and approximately one-third of the Clp for ILT-allo. Clu,s was approximately 2 times greater for ILT-threo than for ILT-allo in dogs, the species for which a carbamoyl glucuronide conjugate was a significant drugrelated component of systemic circulation.
Both ILT-threo and ILT-allo were metabolized primarily to sulfoxide in rats and monkeys, and to sulfoxide and carbamoyl glucuronide in dogs. A sulfone was detected as a minor metabolite. Multiple P450s and FMOs were capable of forming the sulfoxide metabolite, including the extrahepatic FMO1 (intestine), FMO2 (lung), and FMO5 (kidney).
Metabolism to a carbamoyl glucuronide was significant in the dog, accounting for ∼30% of the dose administered for both compounds. It has been reported that carbamoyl glucuronide conjugates are formed by reaction of the amine with carbon dioxide to form a carbamic acid with subsequent conjugation with glucuronic acid. The first step, reaction with CO2, does not require enzymatic catalysis: spontaneous carbamate formation occurs for amino acids (Morrow et al., 1974) and other amines (Greenaway and Whatley 1987; Delbressine et al., 1990). The reversible formation of carbamic acids and their importance in biological systems, such as the mechanism by which hemoglobin carries CO2 from tissues to lungs, is well known (Kilmartin and Rossi-Bernardi, 1973; Weiss and Choi, 1988; Mroz, 1989). The second step involves blocking the dissociation of CO2 from parent drug by glucuronide conjugation, catalyzed by UDP-glucuronosyl transferase in the presence of UDPGA as a cofactor.
Carbamoyl glucuronide metabolites of primary amines are rare. This pathway, which constituted a major metabolic route of tocainide in humans (Elvin et al., 1980), was of quantitative insignificance in preclinical species (Gipple et al., 1982). Carbamoyl glucuronide metabolites of primary amines also were reported for rimantadine (Brown et al., 1990) and for mofegiline, a selective enzyme-activated irreversible inhibitor of monoamine oxidase B (Dow et al., 1994). For mofegiline, 9% of the administered dose was recovered as the N-carbamoyl-O-β-d-glucuronide of parent drug in dog (5 mg/kg i.v. and 20 mg/kg p.o.) and in human urine (24 mg/kg p.o.). Carbamoyl glucuronides of other amines have been reported for the N-desmethyl metabolite of benzazepine (Straub et al., 1988), the carbamic acid ester of the secondary amine of the antidepressant sertraline (Tremaine et al., 1989), the N-desmethyl metabolites of a tetracyclic piperazinobenzazepine and a pyrrolidine (Delbressine et al., 1990), carvedilol (Schaefer, 1992), the N-desmethyl metabolite of a calcium antagonist, Ro 40-5967 (Wiltshire et al., 1992), and the N-dealkylated metabolite of ropinirole (Ramji et al., 1999).
Metabolism of primary amines to carbamoyl glucuronides is a common metabolism route for several structurally diverse DP-IV inhibitors. It is possible that the small number of carbamoyl glucuronides reported in the literature is due to the lack of technology available in the past to properly characterize them. Historically, the presence of glucuronide conjugates was confirmed by hydrolyzing the glucuronide moiety to parent with β-glucuronidase. Under these conditions, the carbamate also reverts to parent and, thus, goes undetected. The possibility that many carbamoyl glucuronides may have been misidentified as N-glucuronides in literature, when this approach was used for identification, cannot be excluded.
In vitro production of a carbamoyl glucuronide at the tertiary amine of carvedilol was demonstrated in dog and rat liver microsomes fortified with UDPGA in the presence of CO2 (Schaefer, 1992). Using similar procedures, in vitro production of the carbamoyl glucuronide conjugates of ILT-threo and ILT-allo was achieved in rat or dog liver microsomes; trace amounts were produced in monkey liver microsomes and none in human liver microsomes. This was accomplished in a CO2-enriched environment in either KH2PO4 or NaHCO3 buffer. The presence of NaHCO3 buffer increased the yield of the carbamoyl glucuronide under our study conditions. Formation of the carbamoyl glucuronide was also favored by the use of alamethicin instead of glycerol/lubrol. Although carbamoyl glucuronides can be produced in vitro in rat and dog liver microsomes in a CO2-enriched environment, they are only a significant biotransformation pathway in vivo in dogs. It is possible that the more efficient renal excretion of ILT-threo in rats may be contributing to the low amounts of carbamoyl glucuronide observed in this species. The reason for this occurrence, however, remains to be investigated.
The unsuitability of the in vitro systems evaluated as predictors of species differences in the metabolism of these DP-IV inhibitors creates a challenge in predicting their metabolism and pharmacokinetics in humans. Thus, dog is the preferred nonrodent species for toxicological evaluation, since it presents a different clearance mechanism from that observed in monkeys and rats.
Acknowledgments
We thank the following individuals, all of Merck Research Laboratories: Dr. Susan Iliff and Bonnie Friscino for assistance with the pharmacokinetic studies in monkeys; Dr. William Feeney, Don Hora, Paul Cunningham, Alison Parlapiano, and Gina Seeburger for assistance in the pharmacokinetic studies in dogs; Jianmei Pang for preparation of the hepatocytes; Dr. Emma Parmee for providing synthetic standards; Dr. Ann Weber for outstanding leadership, thought, stimulating discussions, and assistance in the preparation of the manuscript; and Dr. Stella Vincent and Dr. Ralph Stearns for outstanding mentorship and assistance in the preparation of the manuscript.
Footnotes
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↵1 Abbreviations used are: GLP-1, glucagon-like peptide 1; DP-IV, dipeptidyl peptidase IV; ILT-threo, di-[2S,3S]-2-amino-3-methyl-pentanoic-1,3-thiazolidine fumarate; ILT-allo, the allo stereoisomer of ILT-threo; IS, internal standard; MRL, Merck Research Laboratories; 5-DPT, 10-[(N,N-dimethylamino)-alkyl]-2-(trifluoromethyl)phenothiazine; UDPGA, UDP-glucuronic acid; HPLC, high performance liquid chromatography; P450, cytochrome P450; FMO, flavin-containing monooxygenase; LC-MS/MS, liquid chromatography-tandem mass spectrometry; AUC, area under the plasma concentration versus time curve; Cl, clearance; Ro 40–5967, mibefradil.
- Received May 27, 2003.
- Accepted July 11, 2003.
- The American Society for Pharmacology and Experimental Therapeutics