Drug Metabolism and Disposition Fast Forward
First published on August 14, 2008; DOI: 10.1124/dmd.108.021642
0090-9556/08/3611-2393-2403$20.00
DMD 36:2393-2403, 2008
Preclinical Pharmacokinetics of a HepDirect Prodrug of a Novel Phosphonate-Containing Thyroid Hormone Receptor Agonist
James M. Fujitaki,
Edward E. Cable,
Bruce R. Ito,
Bao-Hong Zhang,
Jinzhao Hou,
Chun Yang,
David A. Bullough,
James L. Ferrero,
Paul D. van Poelje,
David L. Linemeyer, and
Mark D. Erion
Departments of Biological Sciences (J.M.F., E.E.C., B.R.I., B.-H.Z., J.H., P.D.v.P., D.L.L., M.D.E.) and Preclinical Development (C.Y., D.A.B., J.L.F., M.D.E.), Metabasis Therapeutics, Inc., La Jolla, California
(Received March 25, 2008;
Accepted August 12, 2008)
 |
Abstract
|
|---|
The prodrug [(2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane (MB07811)] of a novel phosphonate-containing thyroid hormone receptor agonist [3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxylmethylphosphonic acid (MB07344)] is the first application of the HepDirect liver-targeting approach to a non-nucleotide agent. The disposition of MB07811 was characterized in rat, dog, and monkey to assess its liver specificity, which is essential in limiting the extrahepatic side effects associated with this class of lipid-lowering agents. MB07811 was converted to MB07344 in liver microsomes from all species tested (CLint 1.23-145.4 µl/min/mg). The plasma clearance and volume of distribution of MB07811 matched or exceeded 1 l/h/kg and 3 l/kg, respectively. Although absorption of prodrug was good, its absolute oral bioavailability as measured systemically was low (3-10%), an indication of an extensive hepatic first-pass effect. This effect was confirmed by comparison of systemic exposure levels of MB07811 after portal and jugular vein administration to rats, which demonstrated a hepatic extraction ratio of >0.6 with liver CYP3A-mediated conversion to MB07344 being a major component. The main route of elimination of MB07811 and MB07344 was biliary, with no evidence for enterohepatic recirculation of MB07344. Similar metabolic profiles of MB07811 were obtained in liver microsomes across the species tested. Tissue distribution and whole body autoradiography confirmed that the liver is the major target organ of MB07811 and that conversion to MB07344 was high in the liver relative to that in other tissues. Hepatic first-pass extraction and metabolism of MB07811, coupled with possible selective distribution of MB07811-derived MB07344, led to a high degree of liver targeting of MB07344.
The thyroid hormones (T3 and thyroxine) are important modulators of lipid homeostasis, thermogenesis, and metabolic rate (Yen, 2001
). The effects of the thyroid hormones are widespread and arise from their binding to specific nuclear receptors found in most cell types and tissues (Hulbert, 2000). There are four main isoforms of thyroid hormone receptors (TRs), TR
1, TR2, TRβ1, and TRβ2, which are expressed differentially in tissues (Lazar, 1993). The lipid-lowering activity of thyroid hormones and related analogs as a treatment for hypercholesterolemia and obesity has been demonstrated in animals as well as in humans (Krotkieswki, 2000). However, to date, the development of TR agonists as safe and effective agents to treat hypercholesterolemia and obesity has been hampered mainly by dose-limiting cardiovascular toxicities, resulting in an insufficient safety window (Underwood et al., 1986). Because beneficial effects such as lowering of plasma cholesterol are mediated primarily by activation of the TRβ isoform in liver (Forrest et al., 1996) and effects on cardiac function are mediated by TR in heart (Wikstrom et al., 1998), one approach pursued to improve the therapeutic index of TR agonists has focused on TRβ-selective agents (Ye et al., 2003). This strategy, however, does not circumvent other potentially deleterious effects of TRβ activation in extrahepatic tissues, among which are thyroid hormone axis suppression, muscle wasting, and bone loss (Brenta et al., 2007).
The design of a synthetic TR agonist that specifically targets the liver, the major site of cholesterol metabolism, and reduces or eliminates the deleterious cardiac and other extrahepatic side effects should in theory result in an effective therapeutic agent. In an effort to reach this goal, a TRβ-selective, phosphonate-containing agonist was designed: MB07344 (Ki = 35 and 3 nM at TR and TRβ, respectively) (Erion et al., 2007). Because phosphonates are negatively charged at physiological pH, MB07344 was predicted to have a low volume of distribution and to distribute preferentially to liver, potentially because of efficient uptake by hepatic organic anion transporters (Erion et al., 2005a). To enhance liver targeting of the pharmacological effects of this novel agent, MB07811, the HepDirect prodrug of MB07344, was synthesized (Fig. 1).
View larger version (7K):
[in this window]
[in a new window]
|
FIG. 1. Sequence of conversion of HepDirect MB07811 to the active metabolite MB07344 releasing the glutathione (GSH) by-product MB06588.
|
|
The HepDirect approach enables the delivery of certain phosphate and phosphonate drugs to the liver with high specificity (Erion, 2006; Hecker et al., 2007). By masking the phosphate or phosphonate groups, HepDirect prodrugs are predicted to increase the absorption of the active metabolite by increasing its permeability across the gut membrane (Erion et al., 2004). HepDirect prodrugs are then specifically activated by CYP3A, a P450 enzyme that is localized predominantly in the liver, releasing the active metabolite or active metabolite precursor and a by-product that is neutralized by conjugation with glutathione (Erion et al., 2005b), which is present at millimolar concentrations in liver. Formation of the by-product is not associated with hepatocellular or other toxicity (Erion et al., 2005b). To date, HepDirect prodrugs of 9-(2-phosphonylmethoxyethyl) adenine, i.e., pradefovir (MB06866Q), an oral hepatitis B virus antiviral drug (Lin et al., 2005), and of cytarabine 5'-monophosphate, i.e., MB07133, an agent delivered by i.v. infusion for the treatment of hepatocellular carcinoma, have reached clinical development status (Boyer et al., 2006). Pradefovir and MB07133 are prodrugs of mononucleotide analogs and target their respective therapeutically active metabolites (i.e., nucleoside analog 5'-triphosphates) to the liver in animal models (Erion et al., 2005b). MB07811 is the first non-nucleotide HepDirect prodrug to be evaluated in humans.
Preclinical proof of concept for MB07811 was obtained in multiple studies in normal rodents and rodent models of hyperlipidemia (Erion et al., 2007). The pharmacological targeting of MB07811-derived MB07344 is perhaps best illustrated by an evaluation of the relative effects of MB07811 and T3 treatment of rats on the expression of TR-responsive genes in various tissues. As shown in Table 1, acute treatment with MB07811 resulted in marked changes in expression of TR-responsive genes in liver but relatively minor changes in the other tissues evaluated in contrast with T3 treatment, which resulted in marked changes in gene expression in all tissues evaluated. In repeat dose studies in the rat, doses of MB07811 up to 50 mg/kg/day (
125-fold the ED50 for cholesterol lowering) did not affect heart rate or contractile function (Erion et al., 2007). In contrast, T3 treatment led to marked increases in heart rate and left ventricular pressure at doses <7-fold higher than the ED50 for cholesterol lowering (0.012 mg/kg). These and other studies allowed the conclusion that MB07811 has a significantly improved therapeutic index relative to the natural TR ligand, T3.
View this table:
[in this window]
[in a new window]
|
TABLE 1 Relative mRNA levels in tissues of male Sprague-Dawley rats (n = 6 per group) treated with either MB07811 or T3 at single doses in 10-fold excess of the ED50 of the cholesterol-lowering response in cholesterol-fed rats (4 and 0.12 mg/kg, respectively)
The values represent the fold change at the time point at which a maximal response was obtained with T3 treatment (3-, 8-, and 24-h time points were evaluated). See Erion et al. (2007) for detailed methods.
|
|
Preclinical pharmacokinetic properties of MB07344 and MB07811 were evaluated in the rat, dog, and monkey, species that have served as models for evaluating cholesterol lowering. Studies described here assessed the liver first-pass effect and distribution of MB07344 to test the concept that administration of its HepDirect prodrug leads to liver targeting of the active metabolite.
|
Materials and Methods
|
|---|
Materials. All protocols involving animal experimentation were reviewed and approved by the respective Institution Animal Care and Use Committee of each test facility. Unless otherwise specified, studies were performed at Metabasis Therapeutics, Inc. (La Jolla, CA). In-life procedures were in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). The HepDirect prodrug MB07811, its active metabolite MB07344, and MB06588, the glutathione adduct of the by-product formed after conversion of MB07811, were prepared at Metabasis Therapeutics, Inc. (Fig. 1). Radiolabeled [bridge-14C]MB07811, [5'-3H]MB07811, and [5'-3H]MB07344 were synthesized at Moravek Biochemicals (Brea, CA). Animal and human liver microsomes were purchased from In Vitro Technologies (Baltimore, MD). Microsomes prepared from baculovirus-infected insect cell lines containing wild-type baculovirus (empty vector), recombinant human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2D6, CYP3A4, and CYP3A5 (Supersomes) were purchased from BD Gentest (Woburn, MA). Chemicals were reagent grade or better. Plasma and tissue awaiting analysis were stored frozen at -20°C.
In Vitro Studies. Kinetics of conversion of MB07811 to MB07344 in animal and human liver microsomes and effect of ketoconazole. The conversion of MB07811 to MB07344 in pooled male and female human, cynomolgus monkey, beagle dog, Sprague-Dawley (SD) rat, New Zealand White rabbit, and ICR/CD1 mouse liver microsome preparations was determined by quantification of MB06588, a known by-product of the reaction, and/or MB07344 by reverse-phase high-performance liquid chromatography (HPLC). After verification of the linearity of the reactions, substrate saturation kinetics was determined at a 1 mg/ml microsomal protein concentration and a range of MB07811 concentrations (12.5, 25, 50, 100, and 200 µM) in duplicate reaction mixtures (5 min, 37°C). Reactions were quenched with methanol (60%, v/v). The Enzyme Kinetics module of SigmaPlot software (version 9.01; Systat Software, Inc., Point Richmond, CA) was used to determine Vmax and Km kinetic values by fitting the kinetic data to the Henri-Michaelis-Menten equation. SigmaPlot was used to generate double-reciprocal data plots. CLint values were calculated by dividing Vmax values by Km values. Statistical significance was determined by an unpaired Student's t test using GraphPad InStat software (version 3.01 for Windows 95/NT; GraphPad Software Inc., San Diego, CA), and p < 0.05 was considered statistically significant. Vmax, Km, and CLint values are reported as mean values and S.E.M. To determine whether the conversion of MB07811 to MB07344 is mediated by CYP3A4, the metabolism of MB07811 was assessed in human liver microsomes in the presence of a specific CYP3A4 inhibitor, ketoconazole (0.01, 1, and 100 µM).
Metabolism of MB07811 in liver microsomes. Tritium-labeled [5'-3H]MB07811 was incubated in NADPH- and glutathione-fortified, mixed pools of liver S9 or microsomes (2 mg/ml) from male and female SD rats, male beagle dogs, male cynomolgus monkeys, or male humans for 0 to 1 h at 37°C. The reactions were terminated by addition of 1.5 volumes of methanol. After mixing and centrifugation of the extract to remove the protein, the supernatants were analyzed by on-line radiography-coupled HPLC. Interspecies differences were qualitatively assessed by visual comparison of the radiochromatograms.
CYP phenotyping. MB07811 (5 µM) was incubated in triplicate with microsomes prepared from baculovirus-infected insect cell lines containing recombinant human P450 enzymes (25 pmol), including CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP3A4, and CYP3A5, for 0, 15, and 30 min. Because the protein concentration per picomole of enzyme activity varied among the recombinant enzymes, the protein concentration per incubation was normalized by making up the difference with microsomal protein from wild-type baculovirus-infected insect cells (control cells). The reactions were started by addition of NADPH solution (1 mM) and terminated by addition of ice-cold acetonitrile. Incubations with microsomes prepared from control cells served as a negative control. Conversion of MB07811 to MB07344 was monitored by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The activity (nanomoles of MB07344 per minute per milligram of microsomal protein) was normalized for estimated mean expression levels of each P450 enzyme in the liver (Drahushuk et al., 1998; Dennison et al., 2007; Harper and Brassil, 2008).
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 2. Representative radiochromatograms of [3H]MB07811 and its products in male (A) rat, (B) dog, (C) monkey, and (D) human liver microsomes at 0 (dashed line) and after 30 min (solid line) of incubation.
|
|
Animal Studies. Pharmacokinetics of MB07811 in rats. Three groups (n = 5/group) of instrumented, male, SD rats (250-300 g, 6-8 weeks old) were dosed i.v. at the carotid artery with MB07811 at 3 mg/kg in 100% propylene glycol or p.o. with MB07811 at 3 or 10 mg/kg in 100% polyethylene glycol-400 (PEG-400). Animals were fasted for 3 h before p.o. administration and were refed 1 h after dosing. Animals had free access to food before and during the i.v. evaluation. Blood samples were collected from the jugular vein at predose and at 5, 20, and 40 min and 1, 1.5, 2, 3, 5, 8, 12, and 24 h after i.v. administration of MB07811 or at 0.5, 1, 2, 3, 4, 5, 6, 8, 12, and 24 h after p.o. administration of MB07811. Plasma was prepared from blood samples by centrifugation.
First-pass effect of MB07811 in the rat. Instrumented male SD rats (230-250 g, 6-8 weeks old, n = 7-8) were administered a 2-min i.v. infusion of 1 mg/kg MB07811 as a solution in propylene glycol via either the jugular or portal vein. Blood samples (30-100 µl) were collected from the carotid artery at prespecified times (predose and 0.5, 1, 2, 4, 8, 12, and 24 h postdose), heparinized, and centrifuged to obtain plasma. Plasma samples were extracted with methanol, and the resulting supernatants were analyzed for MB07811 and MB07344 by LC-MS/MS. The area under the curve (AUC) of the plasma concentration-time profiles of MB07811 and MB07344 from the portal vein (AUCpv) and jugular vein (AUCjv) were determined by noncompartmental analysis. Hepatic extraction (EH) of MB07811 was determined from the equation EH = 1 - (AUCpv/AUCjv) (Ward et al., 2001). The fraction (fh) of the i.v. administered drug metabolized in the liver was calculated from AUCs of the MB07344 metabolite after portal vein and i.v. administration of MB07811, as the AUCjv of MB07344 divided by the AUCpv of MB07344 (Kumar et al., 1999).
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 3. Mean ± S.D. formation of MB07344 from MB07811 by a panel of recombinant human P450 enzymes (n = 3). Metabolite formation was measured after a 30-min incubation of MB07811 (5 µM) with baculovirus-insect cell microsomes containing expressed human P450 enzymes (25 pmol). The activity was normalized for estimated expression levels of each P450 enzyme in the liver and is expressed as nanomoles of MB07344 generated per minute per milligram of microsomal protein.
|
|
Assessment of enterohepatic recirculation of MB07344 in the rat. Noncirculating bile duct-cannulated (n = 3) or naive rats were administered an i.v. bolus of 10 mg/kg MB07344. Blood samples from the jugular vein were taken 2.5, 5, 10, and 20 min and 1, 2, 3, 4, 6, 10, and 24 h postdose. Plasma concentrations of MB07344 were evaluated by LC-MS/MS.
Mass balance and tissue distribution in the rat. In the mass balance study, urine (plus cage wash) and feces were collected over a 96-h interval after i.v. bolus administration (via the tail vein) of 2 mg/kg and oral administration (by gavage) of 5 mg/kg 14C-radiolabeled MB07811 to male SD rats (n = 3-4). The urine and cage wash samples were analyzed for radioactivity by liquid scintillation counting without dilution. The fecal samples were extracted with 50% (v/v) acetonitrile in water, decolorized with sodium hypochlorite at 60°C, and then analyzed for radioactivity by liquid scintillation counting. In the tissue distribution study, male SD rats (250 g, 7 weeks of age) were gavaged with 5 mg/kg 14C-radiolabeled MB07811 (1% carboxymethylcellulose suspension, 210 µCi/kg). Sets of rats (n = 3-4/time point) were sacrificed at 3, 8, 24, and 96 h after dose administration, and the following tissues were harvested: cervical lymph nodes, thyroid gland, testes, epididymal fat, urinary bladder, prostate, spleen, pancreas, stomach, mesenteric lymph nodes, small intestine, large intestine, liver, adrenal glands, kidneys, thymus, heart, lungs, bone marrow, quadriceps muscle, eyes, brain, pituitary gland, skin, blood, plasma, and bone (femur). For the stomach, small intestine, and large intestine, the luminal contents and a wash were also collected. The tissues were rinsed in water, dissolved by the addition of Soluene-350, decolorized as necessary, and then analyzed for radioactive content by liquid scintillation counting. Liquid samples (washes, bone marrow, blood, and plasma) were decolorized and analyzed directly by liquid scintillation counting. Separate samples of liver, kidney, epididymal fat, and small and large intestine collected at 3 h postdose were homogenized in 60% acetonitrile for metabolite identification by HPLC radioactivity analysis as described below.
Quantitative whole body autoradiography (QWBA) in the rat. SD rats were administered 5 mg/kg [14C]MB07811 (100 µCi per animal). At 1, 2, 4, 8, 12, 18, 24, and 96 h after administration, the animals were euthanized, and the intact carcass was frozen in a hexane-dry ice bath. The frozen carcass was embedded in 2% carboxymethylcellulose. Cryosectioning (50-µm-thick slices) of the carcass was performed, and the sections were dried. Digital imaging of the radioactivity was accomplished by phosphor imaging or by direct imaging. Tissue concentrations of radioactivity were determined for the following tissues and/or contents: adipose (brown and white), adrenal gland (cortex and medulla), bile (in duct), blood, bone, bone marrow, brain (cerebrum, cerebellum, and medulla), cecum and contents, epididymis, eye (uvea and lens), Harderian gland, heart, kidney cortex and medulla, large intestine (and contents), liver, lung, lymph node, pancreas, pituitary gland, prostate gland, salivary gland, seminal vesicles, skeletal muscle, skin (pigmented and nonpigmented), stomach (gastric mucosa and contents), small intestine (and contents), spleen, spinal cord, testis, thymus, thyroid, and urinary bladder (and contents).
Pharmacokinetics of MB07811 in dogs. A total of three male and three female adult beagle dogs were dosed i.v. with MB07811 in 100% PEG-400 at 3 mg/kg in the fed state or p.o. with MB07811 at 3 mg/kg in 0.5% carboxymethylcellulose-1% Lutrol F68 in the fasted or fed state at MPI Research (Mattawan, MI). Blood samples (heparin) were taken before and at 5, 15, and 30 min and 1, 2, 4, 6, 8, 12, and 24 h after i.v. administration and before and at 0.5, 1, 2, 4, 6, 8, 12, 16, and 24 h after p.o. administration. Plasma was prepared from blood samples by centrifugation.
Pharmacokinetics of MB07811 in monkeys. A total of 12 adult (2-9 years old) cynomolgus monkeys [six males (5.3-6.4 kg) and six females (2.3-3.7 kg)] were randomized into three groups of two males and two females. Animals were fasted for 2 h, and each group was dosed p.o. with MB07811 in PEG-400 at 3, 10, or 30 mg/kg at MPI Research. Food was returned 1 h after MB07811 administration. In a subsequent study, one of the groups was dosed i.v. with MB07811 in PEG-400 at 3 mg/kg. Animals had free access to food before and during the i.v. evaluation. Blood samples were taken before and at 0.5, 1, 2, 4, 8, 12, and 24 h after p.o. administration and before and at 2, 15, and 30 min and 1, 1.5, 2, 3, 5, 8, 12, and 24 h after i.v. administration. Plasma was prepared by centrifugation.
Tissue distribution in the monkey. Three male cynomolgus monkeys were administered 3 mg/kg [14C]MB07811 (100 µCi per animal). One monkey was euthanized at each time of 4, 12, and 24 h postdose with urine collection from each animal up to its termination time. Tissue concentrations of radioactivity were determined for the following tissues: adipose, adrenal glands, blood, heart, kidney, liver, pituitary gland, skeletal muscle, spleen, and thymus. Urine and tissue samples were processed and measured for radioactivity as described for rats.
Analytical and Pharmacokinetic Analyses. LC-MS/MS analysis of MB07811 and MB07344. Plasma and bile samples were analyzed using a liquid chromatograph-tandem mass spectrometer (API 4000; Applied Biosystems, Foster City, CA) equipped with an Agilent 1100 binary pump and a LEAP injector. Plasma and bile samples were extracted with 1.5 volumes of methanol and then centrifuged to remove the precipitate. A 10-µl sample of the supernatant was injected onto a Luna C8 column (5 µm, 2 x 50 mm; Phenomenex, Torrance, CA) fitted with a SecurityGuard C18 guard column (5 µm, 4.0 x 3.0 mm; Phenomenex) and eluted with a gradient from mobile phase A (20 mM N,N-dimethylhexylamine and 10 mM propionic acid in 20% methanol) to B (20 mM N,N-dimethylhexylamine and 10 mM propionic acid in 80% methanol) at a flow rate of 0.3 ml/min (0 min, 60% B; 0-1 min, 60-100% B; 1-6 min, 100% B; 6-6.1 min, 100-60% B; and 6.1-9 min, 60% B). The injector temperature was set at 10°C. Elution times for MB07344 and MB07811 were approximately 4.1 and 5.6 min, respectively. MB07811 and MB07344 were detected using the MS/MS mode (513/63.1 for MB07811 and 363.3/63.1 for MB07344) and quantified by comparison of peak areas to standard curves obtained by spiking known concentrations of the analytes to blank plasma or bile. Linearity was observed from 10 to 3000 ng/ml and calibration curves for MB07811 and MB07344 were generated. The limit of quantitation (LOQ) for both MB07344 and MB07811 was 10 ng/ml. The analytical method met an acceptable percent coefficient of variance of <20% at the lowest standard (typically 10 ng/ml) and of <15% for higher standard concentrations.
HPLC-UV analysis of MB06588. HPLC-UV analysis of MB06588 was performed on an Agilent 1100 series instrument (Agilent Technologies, Palo Alto, CA). Chromatographic separation was accomplished with an Ultrasphere C18 column (5 µm; 4.6 x 250 mm; Beckman Coulter, Fullerton, CA) equipped with an Econosphere C18 guard column (5 µm, 4.6 x 7.5 mm; W. R. Grace and Co., Columbia, MD). The column was equilibrated with 10% acetonitrile in 20 mM potassium phosphate (pH 6.2) and eluted with a linear gradient of 10 to 60% acetonitrile over 10 min at a flow rate of 1.0 ml/min and at a column temperature of 40°C. The UV absorbance was monitored at 245 nm. The retention time of MB06588 was approximately 6.3 min. MB06588 was quantified by comparison with single or multiple point standards diluted in the appropriate matrix.
HPLC radioactivity analysis. Analysis of the S9, microsomal, and cellular extracts was performed on a HP1050 high-performance liquid chromatograph (Hewlett Packard, Palo Alto, CA). The mobile phase was degassed by helium sparging. The samples were analyzed on a Beckman Ultrasphere C18 column (5 µm, 4.6 x 250 mm; Alltech Associates, Deerfield, IL) equipped with an All-Guard cartridge containing an Econosphere C18 (5 µm, 4.6 x 7.5 mm) guard column (Alltech Associates). The column was equilibrated with 20% acetonitrile and 80% 20 mM potassium phosphate (pH 6.2) and eluted with a linear gradient of 20 to 80% acetonitrile over 20 min at a flow rate of 1.5 ml/min. The column temperature was ambient (22°C) and not controlled. The UV absorbance was monitored at 280 nm and radioactivity was monitored by a Radiometric Series A-100 detector (open channel; PerkinElmer Life and Analytical Sciences, Waltham, MA) connected through an Agilent 35900E interface. The ratio of liquid scintillant Ultima-Flo M or Ultima-Flo AP to HPLC eluent was 3:1. The retention times of MB07344 and MB07811 were approximately 8.4 and 20.4 min, respectively. The retention time of MB06588 was approximately 4 min in the UV chromatograms. [3H]MB07811 and [3H]MB07344 were used to confirm the identification of the prodrug and active metabolite, respectively, in the incubation mixtures.
Pharmacokinetic analysis. The temporal profiles of drug concentrations in plasma were analyzed by noncompartmental methods using WinNonlin (version 1.1; Scientific Consulting, Inc., Cary, NC) as defined by Gibaldi and Perrier (1982). Unless otherwise noted, the AUC of the drug concentration in plasma was determined by trapezoidal summation to the last measurable time point. When mean drug concentrations were calculated, values below the LOQ were treated as zero. Consequently, reported means may be below the LOQ. The absolute oral bioavailability of MB07811 was estimated by comparison of the AUC values of the plasma profile of MB07811 after p.o. dosing of prodrug with the AUC values of MB07811 after i.v. administration of prodrug in each individual monkey (Kwan, 1997). Absorption as measured by relative oral bioavailability of MB07811 was determined by dividing the plasma AUC of MB07344 after oral dosing of MB07811 by the plasma AUC of MB07344 after i.v. administration of the prodrug. The metabolic ratio is defined as the ratio of the active metabolite (MB07344) to prodrug (MB07811) and was calculated by dividing the plasma molar AUC0-24h of MB07344 by that of MB07811.
|
Results
|
|---|
Kinetics of Conversion of MB07811 to MB07344 in Animal and Human Liver Microsomes. The conversion of MB07811 to MB07344 was linear with respect to time and protein concentration in microsome preparations from all six species. The Vmax values ranged from 0.16 (male rabbit) to 2.74 (male rat) nmol/mg/min (Table 2). The Km values ranged from 18.8 (male rat) to 127.0 (female rabbit) µM. For the male human, monkey, dog, rat, rabbit, and mouse liver microsome preparations, the CLint values for MB07811 were 4.9, 34.3, 15.3, 145.4, 1.2, and 4.2 µl/min/mg, respectively. For the female human, monkey, dog, rat, rabbit, and mouse liver microsome preparations, the CLint values were 9.8, 23.7, 10.6, 3.6, 5.0, and 6.8 µl/min/mg, respectively. Ketoconazole at 0.1, 1, and 100 µM inhibited the conversion of MB07811 to MB07344 in human liver microsomes by 10, 73, and 100%, respectively.
View this table:
[in this window]
[in a new window]
|
TABLE 2 Summary of the kinetics of MB07811 metabolism in animal and human liver microsome preparations
Metabolism was measured by the quantification of the derivatized byproduct MB06588 as described under Materials and Methods. Values are expressed as mean ± S.E.M. Number of individual microsome sample pooled = n.
|
|
Metabolism of MB07811 in Liver Microsomes. The rate of metabolism of MB07811 in liver microsomes, as measured by the apparent rate of disappearance of the prodrug peak over time, was highest in the male SD rat liver S9 and microsomes and lowest in the male human (Fig. 2). The general pattern of the profile of the major metabolites was similar between the species: MB07344 and an unknown metabolite, eluting approximately 4 min later, were observed in all species, and both increased over time. In addition, an unresolved peak of radioactivity at the solvent front increased over time in all species. The glutathione adduct by-product MB06588 was observed in most of the UV traces of samples from all species. One minor metabolite peak eluting approximately 2 min before the MB07344 peak was detected in human and monkey samples and, to a lesser extent, in dog samples. This peak was not observed in either male or female rat samples. MB07811 was poorly converted to MB07344 in female SD rat liver S9 and microsomes.
P450 Reaction Phenotyping. The contribution of individual P450 enzymes in the conversion of MB07811 to MB07344 was assessed in a panel of 11 human recombinant P450 enzymes (Fig. 3). Linearity of product formation with incubation time was demonstrated for each of the P450 enzymes. The results indicated that CYP1A1, CYP2C8, CYP3A4, and CYP3A5 catalyzed the formation of MB07344 with the highest activity being attributed to CYP3A4. An evaluation of the intrinsic clearance of MB07811 in each of the P450 enzymes would be required to fully determine the relative importance of each of the individual P450 enzymes to the overall oxidative metabolism of MB07811 (Harper and Brassil, 2008).
Pharmacokinetics of MB07811 after i.v. Bolus Administration. The plasma concentration-time profiles of MB07811 and MB07344 in the rat, dog, and monkey are shown in Fig. 4. The pharmacokinetic parameters are summarized in Table 3. The metabolic ratio after i.v. dosing was highest in the rat and monkey (2) and lowest in the dog (0.1). After i.v. administration of 3 mg/kg, the plasma Cmax values of the active metabolite MB07344 were 0.09, 0.01, and 0.63 µg/ml in the rat, dog, and monkey, respectively.
View this table:
[in this window]
[in a new window]
|
TABLE 3 Pharmacokinetic parameters of MB07811 after i.v. and p.o. administration of 3 and 10 mg/kg, respectively, to rats, dogs, and monkeys
Values are expressed as mean ± S.D.
|
|
Pharmacokinetics of MB07811 after Oral Administration. Metabolic ratios of 9, 7, and 24 were observed after p.o. administration of MB07811 to rats, dogs, and monkeys, respectively. These ratios are considerably higher (7-24-fold) than the corresponding values seen after i.v. dosing, an indication of a large first-pass clearance of the prodrug in the liver. The absolute oral bioavailability of MB07811 was <10% in the rat, dog, and monkey (Fig. 4; Table 3). However, uptake of MB07811 from the gut was good based on the extent of absorption of >20% across the species evaluated (Table 3). Plasma Cmax values of MB07344 of at least 18 ng/ml were observed after administration of 10 mg/kg MB07811 to the rat, dog, and monkey.
First-Pass Effect of MB07811 in the Rat. For the rats dosed i.v. via either the portal or jugular vein, the mean ± S.D. plasma AUClast values of systemic MB07811 were 0.14 ± 0.04 and 0.37 ± 0.12 mg · h/l, respectively (Fig. 5). The corresponding plasma AUC values of systemic MB07344 were 0.45 ± 0.18 and 0.54 ± 0.30 mg · h/l. On the basis of these values, the hepatic extraction ratio (EH) for MB07811 was approximately 0.61. The fraction of the i.v. administered prodrug metabolized in the liver (fH) was 1.20.
Assessment of Enterohepatic Recirculation of MB07344 in the Rat. The plasma concentration-time profile of MB07344 after i.v. bolus administration of MB07344 to noncirculating bile-duct cannulated (n = 3) or naive rats is shown in Fig. 6. The plasma AUC of MB07344 was observed to be similar, an indication that MB07344 was not subject to enterohepatic recycling.
View larger version (10K):
[in this window]
[in a new window]
|
FIG. 6. Mean ± S.D. plasma concentration-time profile of MB07344 after i.v. bolus administration of MB07344 to naive and noncirculating bile-duct cannulated rats (n = 3).
|
|
Mass Balance and Tissue Distribution in the Rat. After i.v. bolus administration of [14C]MB07811 in the mass balance study, the mean percentages of 14C-radioactivity excreted in the urine (including washes) and feces were 3 and 95%, respectively, with a total recovery of radioactivity of 98% after 96 h (Table 4). Extraction recoveries of radioactivity were >80% across the species evaluated. After oral administration, the mean percentages of 14C-radioactivity excreted in the urine (including washes) and feces were 2 and 93%, respectively, with a total recovery of radioactivity of 95% after 96 h. After oral administration of [14C]MB07811 in the tissue distribution study, the mean percentages of total radioactivity remaining in the rat (expressed as a percentage of the total dose given) were 72, 7, and <1% at 3, 24, and 96 h, respectively. The total content and concentration of radiolabel in the liver were at minimum 10-fold higher than those for all other tissues, except the gastrointestinal tract throughout the 96-h time period (Table 5). The levels of radioactivity found in the pituitary gland and bone marrow were negligible, and there was no significant accumulation of radioactivity in muscle, heart, brain, epididymal fat, or kidney. After 96 h less than 0.4% of the radiolabel administered remained in the animals. Of the tissues evaluated in the rat, the liver contained the highest percentage of MB07344 (53%) based on total tissue radioactivity. On the other hand, less than 20% of the radioactivity in the intestines was MB07344.
View this table:
[in this window]
[in a new window]
|
TABLE 4 Recovery of radioactivity in mass balance of radiolabel after i.v. bolus and p.o. administration of [14C]MB07811 to male SD rats (n = 4)
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 5 Tissue and fluid concentrations of [14C]MB07811 and metabolites in the rat
Mean ± S.D. concentrations of radiolabel in specific tissues and fluids after oral administration of 5 mg/kg [14C]MB07811 to male SD rats.
|
|
Quantitative Whole Body Autoradiography in the Rat. Most of the radioactivity was associated with the gastrointestinal tract, its contents, and the liver after p.o. administration of [14C]MB07811 (Fig. 7). Specifically, 1 h after administration, most radioactivity was measured in the small intestinal content, stomach content, and liver. In 8 of 23 tissues/organs in which radioactivity was observed, levels were higher than the blood level. At 2, 4, 8, 12, and 18 h postdose, most of the radioactivity continued to be found in the liver and gastrointestinal tract plus contents. By 96 h postdose, most of the radioactivity was eliminated from the animals, but residual radioactivity was still observed in the liver.
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 7. Typical whole body autoradiograms (renal, adrenal, and mid-line plane, left to right) obtained at 2 (rat 1002) and 4 h (rat 1003) after p.o. dosing of a 5 mg/kg dose of [14C]MB07811. Incremental values indicate relative radioactivity concentrations.
|
|
Tissue Distribution in the Monkey. Of the tissues measured, most of the radioactivity was found in the liver of monkeys administered a p.o. dose of [14C]MB07811 (Table 6). No accumulation of radioactivity was detected in the heart, kidney, spleen, adipose, skeletal muscle, or pituitary gland.
View this table:
[in this window]
[in a new window]
|
TABLE 6 Tissue and fluid concentrations of [14C]MB07811 and metabolites in the monkey
Concentrations of radiolabel in specific tissues and fluids after oral administration of 5 mg/kg [14C]MB07811 to male cynomolgus monkeys. Other than blood and plasma, tissues were collected from monkeys 1001, 1002, and 1003 at 4, 12, and 24 h postdose, respectively.
|
|
|
Discussion
|
|---|
MB07811, a HepDirect prodrug of a phosphonate-containing TR agonist MB07344, is absorbed intact and converted in the liver to the active metabolite by CYP3A, a P450 enzyme that is expressed predominantly in liver (Erion et al., 2007). Liver selectivity is achieved as a result of the liver-selective conversion of MB07811 as well as the limited tissue distribution of MB07344 that is postulated to arise from the phosphonic acid, due to its high negative charge, and the inability of these compounds to distribute into most tissues. Because exposure of TR agonists to extrahepatic tissues could potentially lead to safety issues such as tachycardia, bone loss, muscle wasting, and thyroid hormone axis suppression, demonstration of hepatic extraction and liver targeting were critical elements in the preclinical pharmacokinetic assessment of MB07811 and MB07344.
The expected general pharmacokinetic properties of HepDirect liver-targeted prodrugs are 1) high plasma clearance relative to hepatic blood flow, 2) a high volume of distribution relative to body water volume, and 3) good absorption (>20%) from the gut despite 4) poor absolute oral bioavailability (<20%) as assessed by systemic circulation exposure (Erion et al., 2004). The compounds of this novel class of prodrugs are intended to be susceptible to hepatic first-pass effects as they are designed to be readily converted to their active metabolites or active metabolite precursors by liver CYP3A. The hepatic extraction ratio of MB07811 of 0.61 in rat is in close agreement with the 0.55 value obtained by comparing systemic and portal vein concentrations of MB07811 after p.o. administration (Erion et al., 2007). Moderate plasma clearance relative to hepatic blood flow was observed with MB07811with a systemic clearance in the rat and dog estimated to be in excess of 70% of liver blood flow and estimated to be 40% of liver blood flow in the monkey (Davies and Morris, 1993). Consistent with a moderate to high hepatic first pass, the absolute oral bioavailability of plasma MB07811 was <10% in all three species evaluated. The plasma clearance of MB07811 in the rat exceeded hepatic blood flow, which may be attributed to extrahepatic disposition or metabolism.
Although the absolute oral bioavailability of intact MB07811 is low across the species tested, the absorption of the active agent MB07344 from the gut was significantly enhanced when it was delivered as the HepDirect prodrug. The plasma AUC values of MB07344 generated after i.v. administration of MB07811 via jugular (systemic) and portal veins are similar (i.e., fh 1), an indication of negligible extrahepatic metabolism (Kumar et al., 1999) of MB07811. Therefore, the extent of absorption of MB07344 was estimated by comparing the dose-normalized plasma AUC of MB07344 after p.o. and i.v. administration of MB07811. Based on this measurement, extents of absorption of 29, 50, and 32% are observed in rats, dogs, and monkeys, respectively. These values are a considerable improvement over the oral bioavailability of MB07344 (i.e., <10% in the rat) when it was administered as the free phosphonate (Erion et al., 2007).
Another pharmacokinetic outcome of liver sequestration of HepDirect agents is that the volume of distribution of these prodrugs is extremely high. The volume of distribution of MB07811 was observed to be severalfold higher than the volume of body water in animals. As evident from the tissue distribution and QWBA studies, the apparent large volume of distribution is primarily due to the concentration of MB07811 and its metabolites in the liver as opposed to even distribution of the highly permeable prodrug throughout the body. In contrast, the volume of distribution of MB07344, a charged anion at physiological pH values, is relatively low when it is delivered by i.v. bolus administration. Although the volume of distribution of MB07344 is below blood volume, significant partitioning of the free phosphonate to the liver was observed, suggesting involvement of hepatic organic anion transporters (Miyazaki et al., 2004). Some isoforms of organic anion transporters (e.g., OATP2) are predominantly expressed in the liver (Mizuno et al., 2003; Zhou and You, 2007). Liver targeting of MB07344 is achieved by a combination of low extrahepatic tissue distribution and high liver uptake.
Mass balance studies after i.v. bolus and p.o. administration of [14C]MB07811 to rats demonstrated that the major route of elimination is biliary because the majority of radioactivity was found in the feces independent of route of administration. These results are in agreement with biliary excretion studies of MB07344 that indicated quantitative recovery of MB07344 in the bile after i.v. administration to rats (Erion et al., 2007). Biliary excretion is also likely to be a dominant clearance mechanism for MB07811 in monkeys because the renal clearance was low (<7% of dose) as it had been in lower species. Based on the tissue distribution study, more than 90% of the radioactivity was eliminated from rats after 24 h, an observation consistent with the results of a mass balance study in which 92% of the radioactivity was recovered after a similar time period. Consistent with the QWBA results, this finding indicated only residual radioactivity remaining in the liver at 24 h postdose. The results from both the tissue distribution and QWBA studies confirm the utility of the HepDirect approach for liver targeting. In both rat and monkey tissue distribution studies after p.o. administration of [14C]MB07811, the highest concentration of radioactivity in non-gastrointestinal tract tissue was found in liver, where the major metabolite (i.e., >50% of radioactivity in the rat) was MB07344.
High metabolic ratios of MB07344 to MB07811 observed after p.o. administration to animals indicate that conversion of the prodrug to active metabolite occurs in vivo, and this finding was further confirmed in the kinetics studies. MB07811 was readily converted to MB07344 in liver microsome preparations from humans and all three animal species tested. The CLint values in liver microsome preparations from males were highest in rat, followed by monkey, dog, and human. The largest difference in CLint values between the sexes was observed in rat (41-fold lower in females versus males) probably because total CYP3A activity is lower in female rats owing to lack of the expression of the CYP3A2 isoform found in male rats (Lin et al., 2003). Differences in CLint values between the sexes were within 2-fold in the case of human, monkey, and dog liver microsome preparations. The CLint values of MB07811 in male rat and dog liver microsomes were severalfold higher than the corresponding CLint values of pradefovir, which had demonstrated efficacy in both humans and animals (Tillmann, 2007). In human liver microsomes, the CLint values of MB07811 exceeded those of pradefovir (Lin et al., 2005). On the basis of the dose-dependent inhibition of the conversion of MB07811 to MB07344 in human liver microsomes by ketoconazole, a specific inhibitor of CYP3A4 (Turan et al., 2001), CYP3A4 is a major isoform responsible for the conversion of MB07811 although a contribution from other P450 isozymes is possible as inhibition was incomplete at 1 µM ketoconazole. A major role of CYP3A4 was confirmed by monitoring the conversion of MB07811 to MB07344 by a panel of recombinant P450 enzymes. Minor contributions to MB07811 conversion to MB07344 by other hepatic P450 enzymes (CYP2C8 and CYP3A5) and, to an even lesser extent, by a nonhepatic P450 enzyme (CYP1A1) were also observed. A contribution of CYP1A1 is probably minimal on the basis of the liver selectivity observed in the tissue distribution studies.
The metabolic profile of [3H]MB07811 after incubation with liver microsomes was similar among humans and animals. The major metabolites observed by radiography-coupled HPLC after incubation of MB07811 in human and animal liver S9 and microsomes included the active metabolite MB07344, a known byproduct MB06588 (Fig. 1), an unidentified product with a retention time between those of MB07344 and MB07811 and an unresolved peak of radioactivity eluting at the solvent front. An additional minor metabolite (<5% of total radioactivity) was observed in human, monkey, and, to a lesser extent, in dog liver microsomes but not in rat preparations. The metabolic profile of MB07811 in human liver microsomes was most similar to that of monkey liver. Current studies are underway to identify the unknown metabolites and will be reported elsewhere.
In summary, oral absorption of the TR agonist MB07344 is significantly improved as the HepDirect prodrug MB07811. Once in the liver, the prodrug is converted to the active and major metabolite MB07344 by action of CYP3A, resulting in a hepatic first-pass extraction of MB07811 of >60%. Both MB07811 and MB07344 are eliminated in the bile, with some being released from the liver into the general circulation and ultimately returning to the liver to be cleared. Consistent with its low oral bioavailability, MB07344 eliminated in the gut is not reabsorbed nor enterohepatically recirculated. On the basis of the low volume of distribution of MB07344 and confirmation by radiolabeled tissue distribution studies, the circulating TR agonist is not expected to be significantly taken up by extrahepatic tissues such as heart, pituitary, thyroid glands, bone, or muscle, thereby reducing its potential to adversely affect heart rate, contraction, and conductance or thyroid hormone production and metabolism or to cause bone loss or muscle wasting. Like other phosphonates, circulating MB07344 is probably taken into the liver via organic anion transporters because of its negative charge. This property coupled with the characteristics of the HepDirect prodrug leads to enhanced liver targeting and thereby the significant improvement of the therapeutic index observed in preclinical proof-of-concept studies (Erion et al., 2007).
|
Acknowledgments
|
|---|
We acknowledge the contributions of Don Reeder, Bert Chi, Scott Potter, Michael Insko, Xuehong Song, Cindy Phan, and Michael Estes to this manuscript.
|
Footnotes
|
|---|
HepDirect is a registered trademark of Metabasis Therapeutics, Inc.
doi:10.1124/dmd.108.021642.
ABBREVIATIONS: T3, triiodothyronine; TR, thyroid hormone receptor; MB07344, 3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxylmethylphosphonic acid; MB07811, (2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane; MB06866Q, pradefovir, [(2R,4S)-9-[2-[4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphorinan-2-yl]methoxyethyl]adenine; MB07133, (2R,4S)-4-amino-1-[5-O-[2-oxo-4-(4-pyridyl)-1,3,2-dioxaphosphorinan-2-yl]-β-D-arabinofuranosyl]-2(1H)-pyrimidinone; MB06588, L-glutamyl-L-(S-(3-oxo-3-(3-chlorophenyl)propyl)cysteinylglycine; SD, Sprague-Dawley; HPLC, high-performance liquid chromatography; P450, cytochrome P450; LC, liquid chromatography; MS/MS, tandem mass spectrometry; PEG-400, polyethylene glycol 400; AUC, area under the curve; QWBA, quantitative whole body autoradiography; LOQ, limit of quantitation.
Address correspondence to: Dr. James M. Fujitaki, Metabasis Therapeutics, Inc., 11119 North Torrey Pines Rd., La Jolla, CA 92037. E-mail: fujitaki{at}mbasis.com
|
References
|
|---|
Boyer SH, Sun Z, Jiang H, Esterbrook J, Gómez-Galeno JE, Craigo W, Reddy KR, Ugarkar BG, MacKenna DA, and Erion MD (2006) Synthesis and characterization of a novel liver-targeted prodrug of cytosine-1-β-D-arabinofuranoside monophosphate for the treatment of hepatocellular carcinoma. J Med Chem 49: 7711-7720.[CrossRef][Medline]
Brenta G, Danzi S, and Klein I (2007) Potential therapeutic applications of thyroid hormone analogs. Nat Clin Pract Endocrinol Metab 3: 632-640.[CrossRef][Medline]
Davies B and Morris T (1993) Physiological parameters in laboratory animals and humans. Pharm Res 10: 1093-1095.[CrossRef][Medline]
Dennison JB, Jones DR, Renbarger JL, and Hall SD (2007) Effect of CYP3A5 expression on vincristine metabolism with human liver microsomes. J Pharmacol Exp Ther 321: 553-563.[Abstract/Free Full Text]
Drahushuk AT, McGarrigle BP, Larsen KE, Stegeman JJ, and Olson JR (1998) Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture. Carcinogenesis 19: 1361-1368.[Abstract/Free Full Text]
Erion MD (2006) Prodrugs for liver-targeted drug delivery, in Prodrugs: Challenges and Rewards, Part 1 (Stella VJ, Borchardt RT, Hageman M, Oliyai R, Maag H, Tilley J eds) pp 529-560, Springer, New York.
Erion MD, Cable EE, Ito BR, Jiang H, Fujitaki JM, Finn PD, Zhang BH, Hou J, Boyer SH, van Poelje PD, et al. (2007) Targeting thyroid hormone receptor-β agonists to the liver reduces serum cholesterol and hepatic triglycerides and widens the therapeutic index. Proc Natl Acad Sci U S A 104: 15490-15495.[Abstract/Free Full Text]
Erion MD, Reddy KR, Boyer SH, Matelich MC, Gomez-Galeno J, Lemus RH, Ugarkar BG, Colby TJ, Schanzer TJ, and van Poelje PD (2004) Design, synthesis, and characterization of a series of cytochrome P(450)3A-activated prodrugs (HepDirect prodrugs) useful for targeting phosph(on)ate-based drugs to the liver. J Am Chem Soc 126: 5154-5163.[CrossRef][Medline]
Erion MD, van Poelje D, Dang Q, Kasibhatli SR, Potter SC, Reddy MR, Reddy KR, Jiang T, and Lipscomb WN (2005a) MB06322 (CS-917): a potent and selective inhibitor of fructose 1,6-bisphosphatase for controlling gluconeogenesis in type 2 diabetes. Proc Natl Acad Sci U S A 102: 7970-7975.[Abstract/Free Full Text]
Erion MD, van Poelje PD, MacKenna DA, Colby TJ, Montag AC, Fujitaki JM, Linemeyer DL, and Bullough DA (2005b) Liver-targeted drug delivery using HepDirect prodrugs. J Pharmacol Exp Ther 312: 554-560.[Abstract/Free Full Text]
Forrest D, Hanebuth E, Smeyne RJ, Everds N, Stewart CL, Wehner JM, and Curran T (1996) Recessive resistance to thyroid hormone in mice lacking thyroid hormone receptor β: evidence for tissue-specific modulation of receptor function. EMBO J 15: 3006-3015.[Medline]
Gibaldi M and Perrier D (1982) Pharmacokinetics, 2nd ed, Marcel Dekker, New York.
Harper TW and Brassil PJ (2008) Reaction phenotyping: current industry efforts to identify enzymes responsible for metabolizing drug candidates. AAPS J 10: 200-207.[CrossRef][Medline]
Hecker SJ, Reddy KR, van Poelje PD, Sun Z, Huang W, Varkhedkar V, Reddy MV, Fujitaki JM, Olsen DB, Koeplinger KA, et al. (2007) Liver-targeted prodrugs of 2'-C-methyladenosine for therapy of hepatitis C virus infection. J Med Chem 50: 3891-3896.[CrossRef][Medline]
Hulbert AJ (2000) Thyroid hormones and their effects: a new perspective. Biol Rev Camb Philos Soc 75: 519-631.[Medline]
Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals, 7th ed. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington, DC.
Krotkiewski M (2000) Thyroid hormones and treatment of obesity. Int J Obes Relat Metab Disord 24: S116-S119.[CrossRef]
Kumar S, Riggs KW, and Rurak DW (1999) Role of liver and gut in systemic diphenhydramine clearance in adult nonpregnant sheep. Drug Metab Dispos 27: 297-302.[Abstract/Free Full Text]
Kwan KC (1997) Oral bioavailability and first-pass effects. Drug Metab Dispos 25: 1329-1336.[Abstract/Free Full Text]
Lazar MA (1993) Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14: 184-193.[Abstract/Free Full Text]
Lin C-C, Xu C, Teng A, Yeh LT, and Peterson J (2005) Pharmacokinetics of pradefovir and PMEA in healthy volunteers after oral dosing of pradefovir. J Clin Pharmacol 45: 1250-1258.[Abstract/Free Full Text]
Lin G, Cui YY, and Liu XQ (2003) Gender differences in microsomal metabolic activation of hepatotoxic clivorine in the rat. Chem Res Toxicol 16: 768-774.[Medline]
Miyazaki H, Sekine T, and Endou H (2004) The multispecific organic anion transporter family: properties and pharmacological significance. Trends Pharmacol Sci 25: 654-662.[CrossRef][Medline]
Mizuno N, Niwa T, Yotsumoto Y, and Sugiyama Y (2003) Impact of drug transporter studies on drug discovery and development. Pharmacol Rev 55: 425-461.[Abstract/Free Full Text]
Tillmann HL (2007) Pradefovir, a liver-targeted prodrug of adefovir against HBV infection. Curr Opin Investig Drugs 8: 682-690.[Medline]
Turan VK, Mishin VM, and Thomas PE (2001) Clotrimazole is a selective and potent inhibitor of rat cytochrome P450 3A subfamily-related testosterone metabolism. Drug Metab Dispos 29: 837-842.[Abstract/Free Full Text]
Underwood AH, Emmett JC, Ellis D, Flynn SB, Leeson PD, Benson GM, Novelli R, Pearce NJ, and Shah VP (1986) A thyromimetic that decreases plasma cholesterol levels without increasing cardiac activity. Nature 324: 425-429.[CrossRef][Medline]
Ward KW, Proksch JW, Levy MA, and Smith BR (2001) Development of an in vivo preclinical screen model to estimate absorption and bioavailability of xenobiotics. Drug Metab Dispos 29: 82-88.[Abstract/Free Full Text]
Wikström L, Johansson C, Saltó C, Barlow C, Campos-Barros A, Forrest D, Thorén P, and Vennström B (1998) Abnormal heart rate and body temperature in mice lacking thyroid hormone receptor alpha 1. EMBO J 17: 455-461.[CrossRef][Medline]
Ye L, Li YL, Mellström K, Mellin C, Bladh LG, Koehler K, Garg N, Garcia-Collazo AM, Litten C, and Husman B (2003) Thyroid receptor ligands. 1. Agonist ligands selective for the thyroid receptor beta1. J Med Chem 46: 1580-1588.[CrossRef][Medline]
Yen PM (2001) Physiological and molecular basis of thyroid hormone action. Physiol Rev 81: 1097-1142.[Abstract/Free Full Text]
Zhou F and You G (2007) Molecular insights into the structure-function relationship of organic anion transporters OATS. Pharm Res 24: 28-36.[CrossRef][Medline]
![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Top
Abstract
Materials and Methods
) and MB07344 (
) after single p.o. and i.v. bolus administration of 10 mg/kg MB07811 to male SD rats (n = 5) (p.o., A; i.v., D), beagle dogs (n = 6) (p.o., B; i.v., E), and cynomolgus monkeys (n = 4) (p.o., C; i.v., F).
) vein administration of 1 mg/kg MB07811 to male Sprague-Dawley rats (n = 7-8).