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Departments of Drug Metabolism (B.K., M.M., S.B., E.S., D.S., A.J., B.D., G.D.) and Clinical Pharmacology (G.G., T.C., A.N., E.L.), Merck Research Laboratories, Rahway, New Jersey; and Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania (L.W., R.D.)
(Received January 8, 2007; accepted April 11, 2007)
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Abstract |
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90%. The majority of the dose was excreted within 96 h following dosing. Parent compound was the primary radioactive component circulating in plasma, comprising 42 to 72% of the total radioactivity in plasma for up to 12 h. The only other radioactive component detected in plasma was M2, the acyl glucuronic acid conjugate of the parent compound. The major radioactive component in urine was M2, representing 64% of the total radioactivity. Minor metabolites included hydroxylated epimers (M1/M4) and their glucuronic acid conjugates, which occurred in the urine as urea adducts, formed presumably during storage of samples. Fecal radioactivity profiles mainly comprised the parent compound, originating from unabsorbed parent and/or hydrolyzed glucuronic acid conjugate of the parent compound. Therefore, in humans, MK-0524 was eliminated primarily via metabolism to the acyl glucuronic acid conjugate, followed by excretion of the conjugate into bile and eventually into feces.
) that is undergoing preclinical and clinical development for the treatment of niacin-induced flushing. Niacin (nicotinic acid), a member of the vitamin B complex, has been used as a dietary supplement in milligram quantities and as a successful plasma lipid-modifying agent when administered in gram quantities. Niacin decreases plasma concentrations of cholesterol, free fatty acids, and triglycerides in humans, increases plasma high-density lipoprotein cholesterol (Shepherd et al., 1979), and decreases plasma very low-density lipoprotein and low-density lipoprotein cholesterol (Knopp, 1999). Furthermore, studies have shown that niacin can be effectively combined with statins to treat patients with low high-density lipoprotein cholesterol (Rubenfire, 2004; Zhao et al., 2004). The major side effect with niacin therapy is the mild to severe cutaneous flushing experienced by patients that is mediated by vasodilation (Vogt et al., 2006). MK-0524, a potent DP1 antagonist, has an IC50 value of 1.1 nM in a mouse DP1 functional assay (C. Sturino, G. O'Neil, N. Lachance, M. Boyd, C. Berthelette, M. Labelle, L. Li, B. Roy, J. Scheigetz, and N. Tsou, unpublished results) and has been shown to block niacin-induced vasodilation in the mouse by 80% (Cheng et al., 2006). Furthermore, clinical studies have shown the efficacy of MK-0524 in attenuating niacin-induced vasodilation/flushing (Cheng et al., 2006).
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). The in vitro metabolism of MK-0524 in nonclinical species and humans has been described elsewhere (Dean et al., 2007). The major metabolite in hepatocytes from all the species was M2, whereas phase I metabolites (hydroxylated and keto derivatives) collectively comprised a minor component of the metabolic profiles (Dean et al., 2007). |
Materials and Methods |
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The acyl glucuronide of MK-0524 (M2) was prepared by the Labeled Compound Synthesis Group, Drug Metabolism at Merck Research Laboratories. The oxidative metabolites M1, M3, and M4 were prepared by the Medicinal Chemistry Group at Merck Frosst Canada Ltd. (Kirkland, QC, Canada) (Nicoll-Griffith et al., 2007). All the other reagents were of the highest commercial quality available.
Subjects and Dose Administration. The study was conducted at Clinical Pharmacology Associates (Miami, FL). Six healthy male volunteers abstained from food and drink overnight for at least 8 h before dosing. Each subject was administered a single p.o. dose of [14C]MK-0524 as two capsules (total of 40 mg; 202.4 µCi/subject) with 240 ml of water. After dosing, each subject received a standardized lunch and dinner at 4 and 10 h, respectively, and a snack in the afternoon.
Sample Collection. Heparinized blood (12 ml) was collected predose and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 24, 36, 48, 60, 72, 96, 120, 144, and 168 h postdose. Blood samples were centrifuged within 30 min of collection at 3000 rpm at 4°C for 10 min. Plasma was separated and stored as two aliquots at –20°C for qualitative and quantitative analysis. Urine samples voided predose and at specific intervals (0–6, 6–12, 12–24, 24–48, 48–72, 72–96, and 96–120 h) were stored at –20°C. All the fecal samples and wipes were individually packaged during predose and throughout the 7 days postdose. Exhaled CO2 was collected predose (within 2 h of dosing) and at 1 and 4 h postdose. Subjects were instructed to exhale through a one-way valve into scintillation vials containing 4 ml of 1:1 mixture of 1 M hyamine hydroxide solution and ethanol and trace amounts of 1% thymolphthalein (blue indicator). The blue solution turns clear on CO2 collection in 40 to 60 s. Following collection, Scintisafe Gel (Fisher Scientific Co., Pittsburgh, PA) mixture (12 ml) was added to each sample; the samples were then stored in darkness at 4°C.
Sample Preparation. Plasma. Samples were stored at –20°C before extraction for analysis. Previously, studies were conducted to show that the acyl glucuronide metabolite (M2) underwent minimum hydrolysis (0.1%) when plasma samples were stored on ice for up to 6 h (Schwartz et al., 2006). Human plasma was prepared for HPLC/tandem mass spectrometry (MS/MS) analysis by the addition of 9 volumes of acetonitrile containing a stable isotope-labeled internal standard. After mixing and centrifugation, the supernatant was acidified (using an equal volume of dilute formic acid in 40% acetonitrile) to prevent hydrolysis of an acyl glucuronide metabolite found in postdose plasma samples. The sample preparation procedure utilized 96-well plates and was partly automated using a robotic sample processor.
For metabolite profiling, an equal volume (1 ml) of plasma samples collected at 1, 2, 4, and 12 h from each individual was pooled and mixed with 3 ml of acetonitrile/water (2:1). The samples were sonicated for 10 min, vortex-mixed for 10 min, and centrifuged at 3000 rpm for 10 min. The supernatant was removed, and the pellets were resuspended in 3 ml of acetonitrile/water (2:1) by sonicating for 30 min and vortex-mixing for 10 min. The samples were then centrifuged at 3000 rpm for 10 min, and the supernatants were evaporated to dryness under N2. The dried extracts were reconstituted with water/acetonitrile/50% acetic acid (7:2:1, v/v/v).
Urine. For metabolite profiling, 0- to 120-h pools of urine samples were prepared for each subject based on volume, wherein a fixed fraction of the total volume of urine from each interval was pooled. Aliquots (5 ml) from each of these pools were combined. Approximately 100,000 dpm (10 ml) was removed from the urine pool and dried under N2 at 25°C overnight. The residue was reconstituted with water/acetonitrile/50% acetic acid (7:2:1, v/v/v), sonicated for 10 min, centrifuged at 3000 rpm for 10 min, and then analyzed by liquid chromatography/mass spectrometry (LC/MS) with radiometric detection. A representative metabolite profile was generated by analyzing a sample consisting of equal volumes of urine extracts from the six subjects.
For isolation of the urea adducts from urine, 5 to 10 ml of urine was evaporated under N2, and the residue was reconstituted with water/acetonitrile/50% acetic acid (7:2:1, v/v/v), sonicated for 10 min, and centrifuged at 3000 rpm for 10 min. The extracts were analyzed by reversed phase (RP)-HPLC, and the radioactive peaks corresponding to each of the adducts were collected separately. Following evaporation of the solvent under a stream of nitrogen, the samples were reanalyzed using a new column, and the pooled fractions were concentrated and subjected to HPLC and NMR analysis.
Feces. Concentrations of radioactivity and percent of radioactive dose excreted in feces were determined at Covance Laboratories, Inc. (Madison, WI). Briefly, samples were combined by subject at 24-h intervals, and the weight of each combined sample was recorded. A weighed amount of ethanol/water (20:80, v/v) was added to the sample to form an approximate 20% fecal homogenate, and the sample was homogenized using a probe-type homogenizer. Aliquots of the feces homogenate samples were transferred into combustion cones in triplicate and weighed.
For metabolite profiling, aqueous homogenates of the feces collected at 0 to 168 h from each individual were pooled according to homogenate weights. Equal weights from the individual pools were combined across subjects. A 1.5-g aliquot containing 100,000 dpm was precipitated with 5 ml of acetonitrile/isopropyl alcohol (4:1), vortex-mixed, and centrifuged at 3000 rpm for 10 min. The supernatant was removed and evaporated to dryness under N2. The residue was reconstituted with water/isopropyl alcohol/50% acetic acid/acetonitrile (5:3:1:1, v/v/v/v) and analyzed by LC/MS with radiometric detection. A representative metabolite profile was generated by analyzing a sample consisting of equal weights of fecal extracts from six subjects.
Toilet tissue. Toilet tissue samples were combined by subject at 24-h intervals, and a weighed amount of ethanol/water (20:80, v/v) was added.
Quantitative Analysis. Determination of MK-0524 concentration in plasma by LC/MS/MS. The LC/MS system consisted of a Shimadzu LC-10AD VP HPLC pump, a Shimadzu SIL-HTC autosampler, and CTU-10ACVP column heater, and a Waters Symmetry C-8 HPLC column (5.0 cm x 2.1 mm; 3 µm), connected to a 0.5-µm in-line filter, was used. Additionally, a PE SCIEX API 4000 tandem mass spectrometer equipped with a turbo ion spray interface and controlled with Analyst software (PE Sciex, Thornhill, ON, Canada) was used. Samples were analyzed using a column flow rate of 0.3 ml/min mobile phase (50:50 acetonitrile/water with 0.05% formic acid). The column temperature was 35°C, and the injection volume was 5 µl. The MS/MS analysis was performed in the positive ionization mode using multiple reaction monitoring; the precursor-to-product ion transitions were m/z 436 
125 and m/z 440 125 for the detection of parent compound and internal standard, respectively. The standard curve range was 10 to 2500 ng/ml.
Measurement of radioactivity. Aliquots (100–200 µl) of plasma and urine samples were mixed with Scintisafe Gel mixture (Fisher Scientific), and the radioactivity was estimated using a Packard Liquid Scintillation Analyzer (model 1900TR) (PerkinElmer Life and Analytical Sciences, Boston, MA). Air-dried triplicate aliquots of feces homogenates were combusted in a model 307 Sample Oxidizer (PerkinElmer Life and Analytical Sciences), and the resulting 14CO2 was trapped in a mixture of Perma Fluor and Carbo-Sorb. Samples were analyzed for radioactivity in model 2900TR liquid scintillation counter (PerkinElmer Life and Analytical Sciences) for at least 5 min or 100,000 counts. Triplicate-weighed aliquots (0.5 g) of the extracts of toilet tissue were analyzed directly by liquid scintillation counter as above.
Metabolite profiling. The LC/MS system consisted of a Series 200 autosampler and two pumps from PerkinElmer (Norwalk, CT), a Packard radioactivity flow detector, and a Sciex 3000 triple quadrupole mass spectrometer controlled by Analyst software (Sciex, version 1.4, Foster City, CA). The mass spectrometer was operated in the negative ion mode. The source voltage was –3750 V, and the probe temperature was 350°C. A Zorbax SB-phenyl column (4.6 x 250 mm; 5 µm) was used for chromatographic separation. Column elution was achieved with a mixture of 10 mM aqueous ammonium acetate containing 0.1% acetic acid (mobile phase A) and acetonitrile/methanol (92.8: 7.2, v/v) containing 7.2 mM ammonium acetate and 0.1% acetic acid (mobile phase B). The column was eluted over a 40-min period with a linear gradient from 25 to 65% B. The effluent from the HPLC, pumped at a rate of 1.0 ml/min, was diverted at a 24:1 ratio to the radiometric flow detector and the mass spectrometer, respectively. Scintillation mixture (Packard Ultima Flo-M, Downers Grove, IL) was pumped at a rate of 3 ml/min into the radiometric detector. Contributions of parent compound and metabolites were calculated from the amount of radioactivity eluting in each peak relative to the total radioactivity in the HPLC radiochromatogram. Metabolites were identified by extraction of expected m/z from Q1 TIC scan and by comparison of their retention times and MS/MS spectra with those of available authentic standards.
In vitro formation of urea adducts of M1 and M4. M1 and/or M4 synthetic standards (5 µM) were incubated at room temperature with predose human urine for 5 days or with 2.5 M urea for 24 h. Acetonitrile extracts were analyzed by RP-HPLC coupled with LC/MS/MS.
NMR analysis of urea adducts of M1 and M4. Proton NMR spectra of the parent and metabolites were obtained in acetonitrile-d3/D2O (8:1, by volume) at room temperature using a Varian Inova 600-MHz NMR spectrometer (Varian, Inc., Palo Alto, CA) equipped with a 3-mm Nalorac probe (Varian, Inc.).
Pharmacokinetics. The apparent terminal rate constant (
) for both radioactivity and MK-0524 plasma concentrations was estimated by regression of the terminal log-linear portion of the concentration-time profile (using quantifiable concentrations only); t1/2 was calculated as the quotient of ln(2) and . Onset of the terminal log-linear phase was determined by inspection. Area under the curve (AUC) to the last time point with a detectable plasma concentration (AUC0-last) was calculated using the linear trapezoidal method for ascending concentrations and the log trapezoidal method for descending concentrations. AUC0-
was estimated as the sum of AUC0-last and the extrapolated area given by the quotient of the last measured concentration and . The Cmax and Tmax values were obtained by inspection of the plasma concentration data.
Dose Recovery Calculations. The percentage of radioactive dose recovered was determined by the sum of the percentages excreted in urine, feces, and exhaled air. The percentage of the radioactive dose recovered as metabolites was determined by the product of the percentages of the radioactivity dose recovered and those accounted by metabolites in metabolite profiles.
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Results |
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22% of the administered dose was recovered in the urine within 5 days (range, 17–30%) in the six subjects who participated in this study. Fecal excretion averaged 68% of the administered dose (range, 59–74%) within 7 days, with a mean total recovery of radioactivity in urine and feces of 90% (range, 87–91%). The majority of the dose was excreted within 96 h following dosing. Exhaled air was collected 1 and 4 h postdose and examined for [14C]CO2. Average radioactivity levels at these time points were not substantially different from background levels.
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Plasma Concentrations. The mean concentration profiles of MK-0524 and radioactivity in plasma following administration of a single p.o. dose of [14C]MK-0524 are shown in Fig. 2. Individual plasma MK-0524 and radioactivity pharmacokinetic parameters are listed in Table 2. MK-0524 accounted for 29% of the radioactivity in plasma, as determined by the AUC ratio of MK-0524 to radioactivity. Following p.o. administration, [14C]MK-0524 was absorbed rapidly; the highest MK-0524 and total radioactivity concentrations in plasma (Cmax) were achieved, on average, between 1 to 1.5 h and ranged from 1.27 to 3.59 µM Eq (mean value, 2.34 µM Eq) and 3.60 and 6.44 µM (mean value, 5.07 µM), respectively.
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Identification of Metabolites. Figure 1 shows the structures of metabolites identified in human samples following p.o. administration of [14C]MK-0524. Most of the metabolite identification was done by LC/MS analysis and/or by comparison of their retention times and MS/MS spectra with those of available authentic standards. Detailed LC/MS/MS information on the fragmentation of the metabolites has been described elsewhere (Dean et al., 2007). Two new unknowns were detected in human urine and are discussed below.
[14C]MK-0524 Metabolite Profiles in Plasma. Metabolite profiles in plasma pooled across subjects at 1, 2, 4, and 12 h are presented in Fig. 3. Because of the low levels of radioactivity and the limited volume of plasma, it was not possible to obtain individual subject metabolite profiles. The major circulating components in human plasma were the parent compound, MK-0524, and the acyl glucuronide of the parent (M2). The parent compound represented 42 to 72% of the radioactivity in plasma up to 12 h, whereas M2 comprised 28 to 56% of the radioactivity.
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5% of the radioactivity, whereas M2 was the major component and comprised 64% of the radioactivity in human urine. Minor amounts of the acyl migrated products were also observed. Two new unknowns were observed at 18.40 and 19.70 min in the radiochromatograms of the pooled human urine (Fig. 4). These unknowns gave deprotonated molecular ion [M-H-] at m/z of 492, corresponding to an addition of 58 Da to the parent compound (m/z 434). Further fragment ions were at m/z 449, corresponding to the loss of a CO-NH2 group. The 1H NMR spectra of the m/z 492 adducts revealed the loss of one proton from the methylene adjacent to the indole moiety (i', see Fig. 5) and a 2.5-ppm downfield shift of the remaining proton (i). The chemical shift of i (5.08 ppm) relative to that in the spectrum of the hydroxylated metabolite (5.15 ppm, spectrum not shown) indicated a weaker electron withdrawing group, such as an amide nitrogen. Given the molecular weight and the prevalence of urea in urine, these data suggested a urea adduct. These urea adducts could have formed ex vivo during the storage of urine samples. HPLC analysis of an incubation mixture of a synthetic standard of M1 (m/z 450) with predose urine for 5 days showed that both M1 and its epimerized product M4 were converted to their urea adducts (m/z 492) (Fig. 6A). Similarly, urea adducts of both M1 and M4 were formed following an overnight incubation of M4 in the presence of 2.5 M urea (Fig. 6B). Both incubations resulted in the formation of m/z 492 adducts that yielded fragment ions and 1H NMR spectra identical to the adducts originally isolated from human urine. Urea adducts of the glucuronide conjugates of M1 and M4 gave an m/z of 668. The keto derivative, M3, was also a minor component in human urine (2% of the total radioactivity). Thus, the radioactivity in urine comprised 5% parent compound, 31% phase I (oxidative) metabolites, and 64% phase II (glucuronide conjugate) metabolites.
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Discussion |
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90%. The excretion pattern was similar in all six subjects.
Absorption was rapid, with peak plasma concentrations of total radioactivity and parent compound being achieved within 1.5 h. A comparison of AUC values of the parent compound versus total radioactivity (10.8 versus 35.1 µM/h) indicates that MK-0524 contributed to 30% of the circulating levels of drug-related material in plasma. Plasma radioactivity profiles at select time points (1, 2, 4, and 12 h) revealed that the only other radioactive component, besides MK-0524, was the acyl glucuronic acid conjugate of the parent compound (M2), comprising 28 to 58% of the plasma radioactivity. A similar plasma profile was also observed in dogs dosed p.o. and i.v. with [14C]MK-0524, wherein the relative contributions of the parent compound and M2 were 63 to 88% and 24 to 37%, respectively (Chang et al., 2007). The relative ratio of the concentrations of M2 to MK-0524 in vivo was species-specific in animal studies. Following p.o. and i.v. administration of MK-0524 to rats, dogs, and cynomolgus monkeys, plasma AUC values of M2 were 1%, 10 to 20%, and 29 to 127%, respectively, of those of MK-0524. The stability of M2 in plasma was studied previously (Chang et al., 2007). Briefly, when incubated with fresh plasma from rat, dog, and human, M2 underwent acyl migration to form transient isomeric products and finally hydrolyzed to form the parent compound. The in vitro half-life for M2 hydrolysis in plasma ranged from 1 to 3 h in these species. In this study, human plasma collection methods were optimized and validated to prevent hydrolysis of M2. Studies were conducted to show that the relative extent of hydrolysis of M2 was 0.1% when plasma samples were stored on ice for 6 h (Schwartz et al., 2006). Glucuronidation has been considered as the most common phase II reaction and is mainly a mechanism of detoxification and elimination for a wide variety of endogenous substrates and xenobiotics in mammals. Acyl glucuronides are usually susceptible to hydrolysis by ß-glucuronidases present in the gut, nonspecific esterases, serum albumin, and hydroxide ion (Koster et al., 1985; Bailey and Dickinson, 2003).
Following p.o. administration of MK-0524, about 22% of the administered radioactive dose was excreted in the urine. The major component in urine samples was M2, the acyl glucuronide, comprising 64% of the total radioactivity, along with urea adducts of the hydroxylated epimers (M1/M4) and of their corresponding glucuronides. In addition, M3 (the keto metabolite) was a minor component in human urine. Detailed in vitro studies conducted following incubation of M1 and M4 in the presence of urea and/or control human urine indicated that these urea adducts could have formed in vitro during the storage of study samples and appear to be chemical artifacts arising from M1/M4. Standards of M1 and M4 were found to readily epimerize when analyzed with LC/MS and formed urea adducts when allowed to stand in blank urine or 2.5 M urea. Based on these observations, a plausible mechanism for the formation of the urea adducts involves the acid-catalyzed elimination of water to form a resonance-stabilized carbocation. This carbocation can be trapped by urea to form both urea adduct epimers or water to regenerate the epimers M1/M4.
Approximately 68% of the dose was excreted into the feces following p.o. administration of MK-0524. The parent compound was the major component in human feces accounting for 73% of the total radioactivity excreted via this route. The presence of parent compound in feces presumably represents hydrolyzed M2, acyl glucuronic acid conjugate of the parent compound, along with contribution from the unabsorbed parent compound. Based on unpublished observations, the oral bioavailability of MK-0524 in human subjects was determined to be 80%, which was similar to what was observed in dogs (Chang et al., 2007). Therefore, most of the parent compound present in feces is likely the result of enzymatic hydrolysis of M2, which suggests that MK-0524 was eliminated primarily by metabolism via glucuronidation and further excretion of the acyl glucuronide into bile and feces. In vitro studies also showed that MK-0524 underwent glucuronidation in incubations of human intestinal microsomes conducted in the presence of UDP-glucuronic acid (Dean et al., 2007). Therefore, it is likely that a portion of the p.o. dose of the compound may undergo first-pass glucuronidation in the gut as well during the absorption process. M2 was also the major metabolite formed in vitro in human hepatocyte incubations with MK-0524, and several recombinant human UDP glucuronosyltransferase enzymes (UGT1A1, UGT1A3, UGT1A9, and UGT2B7) could catalyze the acyl glucuronidation of MK-0524 (Dean et al., 2007).
Thus, the routes of elimination of MK-0524 in humans appear to be similar to those in preclinical species. In rats and dogs, MK-0524 was cleared via metabolism exclusively by way of glucuronidation, followed by excretion of the acyl glucuronide (M2) into bile, which represented >95% of the total radioactivity (Chang et al., 2007). Trace metabolites in rat and dog bile comprised the hydroxylated epimers M1/M4 and the keto derivative, M3, along with the corresponding glucuronides of these entities (1–2% of radioactivity). In comparison, human feces profiles showed that M1/M4 and M3 collectively comprised 27% of the radioactivity in a 0- to 168-h feces profile, thus showing that in humans, a significant portion of the dose was eliminated by phase I metabolism. Similarly, human urine profiles also showed that M1/M4, along with their glucuronidated derivatives and M3, accounted for a total of 30% of radioactivity. In vitro studies to identify the cytochrome P450 isoforms involved in the oxidative metabolism of MK-0524 have shown that CYP3A4 was the primary isoform catalyzing the formation of M1/M4 and M3 in human liver microsomes.
In summary, following a p.o. dose of 40 mg/202 µCi, MK-0524 was eliminated primarily by metabolism via formation of M2, the acyl glucuronide, which was further excreted into the bile. The major circulating entity in human plasma following a p.o. dose of MK-0524 was the parent compound along with M2, the acyl glucuronic acid conjugate of the parent compound.
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ABBREVIATIONS: MK-0524, [(3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopentaindol-3-yl]acetic acid; DP1, prostaglandin D2 receptor 1; HPLC, high-performance liquid chromatography; MS/MS, tandem mass spectrometry; LC/MS, liquid chromatography/mass spectrometry; RP, reversed phase; AUC, area under the curve.
Address correspondence to: Bindhu Karanam, Merck Research Laboratories, Department of Drug Metabolism, RY80E-200, P.O. Box 2000, Rahway, NJ 07065. E-mail: bindhu_karanam{at}merck.com
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