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Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania (P.S., M.J.R., J.E.B., J.K.A., M.P.B., P.G.P., T.A.B.) and Rahway, New Jersey (M.A.W.); and Center for Accelerator Mass Spectrometry and Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California (J.S.V., E.A.U., P.T.H.)
(Received May 6, 2004; accepted July 30, 2004)
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Abstract |
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; Beck et al., 2001; Keith-Roach et al., 2001; Mironov et al., 2002), to archaeology and volcanology (Stafford et al., 1984; Vogel et al., 1990; Smith et al., 1999), to its use as a bioanalytical tool for nutritional research (Buchholz et al., 1999; Deuker et al., 2000; Weaver and Liebman, 2002). Biomedical applications of AMS and its use in the arena of pharmaceutical research also have been detailed in review articles (Barker and Garner, 1999; Garner, 2000; Turteltaub and Vogel, 2000). To date, most studies on the metabolism and disposition of xenobiotics by AMS have focused on the binding of carcinogens to DNA and proteins (Turteltaub et al., 1990, 1997; Frantz et al., 1995; Dingley et al., 1999; Li et al., 2003). These studies have demonstrated a linear relationship between dose and DNA adduct formation. Applications of AMS to the field of pharmaceutical sciences have been limited to only a few studies (Kaye et al., 1997; Young et al., 2001; Garner et al., 2002). However, the pharmaceutical industry is becoming increasingly aware of the potential benefits that may accrue from the ultra-high sensitivity afforded by AMS in terms of evaluating the pharmacokinetics of lead drug candidates in early development. Specifically, AMS allows administration of subpharmacological doses (microdoses) of carbon-14 or tritium-labeled investigational drugs to animals or humans at radiologically insignificant levels with the goal of obtaining preliminary information regarding the absorption, distribution, metabolism, and excretion of test compounds (Turteltaub and Vogel, 2000). An unresolved issue, however, is whether the pharmacokinetics determined following a microdose are representative of those following a conventional (pharmacological) dose (Lappin and Garner, 2003). This paper examines the linearity of kinetics of an antiviral nucleoside analog, 7-deaza-2'-C-methyl-adenosine (Compound A), across subpharmacological and pharmacological dose ranges in the dog before initiation of a human microdose study. The present study thus provides the first description of the full pharmacokinetic profile of a drug candidate assessed under these two dosing regimens.
The specific objectives of this study, therefore, were 1) to assess the pharmacokinetics of Compound A in dogs by a conventional dosing approach using LC-MS/MS for sample analysis, 2) to assess the pharmacokinetics of Compound A in dogs by the microdose approach using AMS for sample analysis, 3) to compare the pharmacokinetics of Compound A at a microdose versus a pharmacological dose, and 4) to validate AMS for this application and to compare the sensitivity of AMS to that of LC-MS/MS.
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Materials and Methods |
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Animal Facility for the AMS Study. A room in the Merck Research Laboratories Drug Metabolism animal facility was designated for the purpose of conducting this AMS study in dogs. The room had not been used for any studies involving radiolabeled compounds in the past 6 years. The walls and dog cages were cleaned with detergent numerous times to remove any traces of 14C from previous uses. Small glass fiber filters measuring 2.4 mm in diameter (Fisher Scientific, Pittsburgh, PA) were wet with alcohol and swiped over small areas of interest for monitoring background 14C. Several such swipes were taken of the dog cages, food bowls, water supply, fume hood, air vents, door handles, and bench top surfaces. These were sent to Lawrence Livermore National Laboratory (Livermore, CA) for analysis to certify the absence of background 14C contamination in the room and the environment in which the AMS study was to be performed. The results from these swipe test samples indicated that the environment was suitable for the conduct of an AMS study. No significant areas of contamination were identified.
All procedures for the study were carried out with utmost care to avoid contamination of samples with external sources of 14C. Disposable supplies were used for all sample preparation and handling steps. In general, extreme care was exercised in setting up this facility to assure the successful conduct of the AMS component of the study (Buchholz et al., 2000). Moreover, access to this facility was limited to those researchers directly involved in the study.
Pharmacokinetic Studies. All animal studies described in this paper were approved by the Merck Research Laboratories Institutional Animal Care and Use Committee. Male Beagle dogs from Marshall Farms (North Rose, NY) weighing 8.3 to 11.2 kg were used for the pharmacokinetic studies. Dogs were fasted overnight before drug administration and fed 4 h postdose. The oral dose solutions were prepared in 0.5% aqueous methylcellulose and the intravenous dose solutions were formulated in saline. Administration of the intravenous dose was via the cephalic vein as a slow bolus over 30 s at 0.1 ml/kg, and that of the oral dose solution was by gavage. Three groups of dogs (n = 2/group) were dosed with [14C]Compound A. The first group of dogs was administered a 1 mg/kg oral dose (specific activity 0.017 µCi/mg). The second group of dogs was administered a 0.02 mg/kg oral dose (specific activity 0.546 µCi/mg), and the third group of dogs received a 0.02 mg/kg intravenous dose (specific activity 0.428 µCi/mg). Blood samples were collected from the jugular vein at predose and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 28, 32, 36, 48, 56, 72, and 80 h after drug administration. An additional sample at 0.083 h (5 min) was collected after intravenous administration of the compound.
In a separate pharmacokinetic study, dogs (n = 4, crossover study design) were dosed with unlabeled Compound A. For the first leg of this study, the dogs were administered a 0.4 mg/kg intravenous dose. Two weeks later, the dogs were administered a 1 mg/kg oral dose. Blood samples were collected from the jugular vein at predose and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 28, 32, 48, and 72 h after drug administration. An additional sample was collected at 0.083 h (5 min) after intravenous administration. All blood samples were collected in EDTA tubes and plasma was harvested by centrifugation. All samples were stored at -20°C or -70°C until analysis.
LC-MS/MS of Compound A in Plasma. To 50 µl of plasma was added 50 µl of methanol/water (50:50; v/v), 25 µl of internal standard (0.4 µg/ml tubercidin), and 1 ml of 0.1 M sodium phosphate solution (pH 2.0). The contents were briefly vortexed and the analytes (Compound A and internal standard) were extracted from plasma samples using an Oasis MCX (30-mg) extraction plate (Waters, Milford, MA). The plate was rinsed with 0.5% formic acid (0.5 ml) followed by acetonitrile (0.5 ml), and the analytes were eluted with 300 µl of acetonitrile/water/ammonium hydroxide (90:8:2; v/v). The eluent was neutralized with acetic acid and aliquots (20 µl) injected onto the LC-MS/MS system. Concentrations of parent (Compound A) were determined from a standard curve prepared daily by adding stock working standards of drug and internal standard into 50 µl of drug-free dog plasma. The validated assay demonstrated good linearity and reproducibility for Compound A over the concentration range 2 to 2000 ng/ml. The lower limit of quantitation was 2 ng/ml. The values for assay accuracy and precision (% CV) ranged from 95.5 to 102.3% and 2.1 to 7.2%, respectively. Compound A was shown to be stable in dog plasma during the time required for storage and analysis of the samples and for up to three freeze-thaw cycles (freezing at -20°C and thawing at room temperature).
The HPLC system consisted of a PerkinElmer LC200 pump (PerkinElmer Life and Analytical Sciences, Boston, MA) and a Varian Prostar 430 autosampler (Varian, Inc., Palo Alto, CA). The mass spectrometer was a PE Sciex API 4000 triple quadrupole system (PerkinElmerSciex Instruments, Boston, MA) equipped with a heated nebulizer interface (500°C) operated in the positive ion electrospray mode. Selected reaction monitoring of the precursor-to-product ion pairs m/z 281
135 for Compound A and m/z 267135 for tubercidin was used for quantitation. PE Sciex Analyst Version 1.1 software was used to collect and process the data. The mobile phase consisted of acetonitrile/10 mM ammonium acetate (9:1; v/v), adjusted to pH 4.0 with acetic acid, at a flow rate of 400 µl/min. Chromatography was performed on a 2.0 x 100 mm, 5-µm Tosoh Biosep TSK-GEL Amide-80 column with a 2.0 x 10 mm guard cartridge of the same packing (Tosoh Bioscience LLC, Montgomeryville, PA).
AMS Measurement of Total 14C in Plasma. All biological samples were shipped from Merck Research Laboratories to the Lawrence Livermore National Laboratory on dry ice. Upon receipt, samples were unpacked, inspected, and stored frozen (-20°C) pending analysis. Samples were converted to elemental carbon for AMS quantitation of 14C in a two-stage process. First, samples were oxidized to CO2 in individual sealed tubes containing cupric oxide, followed by reduction onto approximately 10 mg of iron catalyst in a second tube over zinc and titanium hydride (Vogel, 1992; Ognibene et al.,2003). This process requires approximately 400 µg of carbon, corresponding to the amount available in approximately 10 µl of plasma. Aliquots were obtained from the defrosted plasma samples and placed in quartz combustion tubes. The exact sample mass in this process is not important, since AMS measures an isotope ratio that is converted to equivalent amounts of drug per milliliter or milligram of sample using the known isotopic content of the labeled compound and the average or individual carbon content of each sample (Vogel et al., 2001).
Reduced samples were pressed into aluminum holders, mounted in a wheel containing up to 58 samples of which 6 represented standards with well defined isotopic compositions, and analyzed by the 1-mV AMS spectrometer at Lawrence Livermore National Laboratory (Ognibene et al., 2002). Each sample was measured to >15,000 14C counts at least three times and, if necessary, up to seven times such that the last three measurements agreed to within 3% of each other without monotonic trend. The reported isotope ratio was derived by comparison of the measured isotope ratio to that of the known standards. Results were reported in the unit "Modern," which is equivalent to 97.8 attomole of 14C per mg of carbon or 6.11 fCi of 14C per mg of carbon or 0.0136 dpm per mg of carbon. "Modern" represents the expected natural level of 14C in the biosphere and is a National Institute of Standards and Technology-traceable quantity (National Institute of Standards and Technology standard reference material 4990). AMS-derived 14C concentrations, which include parent compound as well as metabolites, were converted to nanogram equivalents of Compound A per milliliter (ng Eq/ml) using the specific activity of the dosed compound.
Pharmacokinetic Analysis. Plasma concentrations of Compound A parent levels (measured by LC-MS/MS) and total 14C levels (measured by AMS) were used to estimate pharmacokinetic parameters for each treatment group and subject. The areas under the plasma concentration-time curve (AUC0-t) were calculated from the first time point up to the last time point with measurable drug concentration using the linear trapezoidal or linear/log-linear trapezoidal rule. The remaining area under the plasma concentration-time curve was estimated by dividing the observed concentration at the last time point by the elimination rate constant, and this value was used to estimate the AUC0-inf. The percentage AUC extrapolated (% AUC Extrap) was a function of (AUC0-inf - AUC0-t) 
100/AUC0-inf. The intravenous plasma clearance was calculated by dividing the dose by AUC0-inf. The alpha, beta, and gamma half-lives (t1/2
, t1/2ß, and t1/2
, respectively) were determined by fitting the plasma concentration-time data to a triexponential decay model with intravenous bolus input and linear first-order elimination from the central compartment using iterative unweighted nonlinear least-squares regression analysis. The regression lines through the plots of observed versus predicted concentrations did not differ from the line of identity, and no bias was observed. The volume of distribution at steady state (Vdss) was obtained from the product of the plasma clearance and the mean residence time. The maximum plasma concentration (Cmax) and the time at which maximum concentration occurred (Tmax) were obtained by inspection of the plasma concentration-time data. Absolute bioavailability was determined from dose-adjusted (AUC0-inf) ratios.
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Results |
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) (17.5 ± 0.7 h). The Vdss was large (8.6 ± 2.4 l/kg) and exceeded total body water. After oral dosing, the absolute bioavailability of Compound A was high (103 ± 5%), with a Tmax and Cmax of 0.44 ± 0.38 h and 849 ± 168 ng/ml, respectively. For comparison, the average pharmacokinetic parameters of Compound A as determined by AMS and derived from a separate study in which radiolabeled compound was administered to dogs are presented in Table 2. These AMS-derived parameters when dose-normalized were comparable to the pharmacokinetic properties obtained for Compound A following analysis by LC-MS/MS (see Table 1).
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Plasma concentration versus time profiles for Compound A after a single 0.02 mg/kg intravenous bolus or oral dose were determined by AMS (total 14C levels) as well as by LC-MS/MS (parent levels) and are presented in Figs. 2 and 3. It should be noted that due to the small dose administered to dogs, it was not possible to quantify plasma levels beyond 2 h postdose by LC-MS/MS, because the levels had dropped below the lower limit of quantitation (2 ng/ml). The much more sensitive technique of AMS permitted monitoring of total 14C plasma concentrations of Compound A down to 0.2 ng/ml, from which a pharmacokinetic profile at the 0.02 mg/kg microdose was obtained.
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The plasma concentration-time profiles of total 14C determined by AMS versus parent levels of Compound A determined by LC-MS/MS after a single 1 mg/kg oral dose are presented in Fig. 4, and the average pharmacokinetic parameters are summarized in Table 3 along with those of the 0.02 mg/kg oral dose for comparison. At a dose of 1 mg/kg, the AUC0-t and Cmax ratios of parent Compound A to total 14C levels were 0.77 and 0.76, respectively, indicating that the parent compound accounted for the majority of the 14C in plasma. The AUC0-t and Cmax at 1 mg/kg as determined by AMS were 2846 ng Eq h/ml and 659 ng Eq/ml, respectively. At the 50-fold lower dose of 0.02 mg/kg, the AUC0-t and Cmax as determined by AMS were proportionately lower (54.5 ng Eq h/ml and 12.7 ng Eq/ml, respectively), i.e., 2725 ng Eq h/ml and 635 ng Eq/ml, respectively, when dose-normalized for a dose of 1 mg/kg. Due to the low levels of radioactivity used in this study, it was not possible to detect total 14C levels in plasma by the conventional technique of LSC. Once again, the enhanced sensitivity of AMS allowed monitoring of these plasma samples at both the pharmacological and microdose levels.
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Discussion |
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). However, an underlying assumption with this approach is that the pharmacokinetics at the microdose (subpharmacological dose) reflect those at the pharmacological dose. This is especially important for compounds that are retained in a target tissue, such as anti-osteoporotics in the bone (Stepensky et al., 2003). In situations where the target tissue cannot be directly sampled, disposition properties of compounds can be estimated from the long-term kinetics of elimination through plasma or excreta (Gregory et al., 1998). Pharmacokinetic studies at subpharmacological doses, therefore, need to utilize analytical procedures that can define not only the initial absorption and distribution phases accurately, but also any slow elimination phase during which analyte concentrations may be very low. AMS has extremely high sensitivity for tracing isotopically labeled compounds, uses small sample sizes (5–10 µl), and can provide detailed kinetic profiles at low drug concentrations.
Preliminary pharmacokinetics of Compound A in dogs have been recently described by our colleagues (Olsen et al., 2004); however, the full pharmacokinetic profile outlining the multiphasic kinetics has not been previously reported. Furthermore, currently there are no published comparisons of the kinetics of a pharmaceutical compound at pharmacological versus subpharmacological doses using microdosing strategies. The present study thus provides the first full description of the pharmacokinetics of a development candidate assessed under these two dosing regimens and demonstrated that, in the case of Compound A, the pharmacokinetics in the dog appear to be linear over a 50-fold dose range. These pilot studies in dogs provide some reassurance that microdose studies with Compound A in humans would provide meaningful predictions of kinetics at higher (pharmacological) doses. From a methodological standpoint, microdose/AMS studies are labor-intensive with respect to sample collection and handling due to the need to prevent contamination by extraneous sources of 14C. However, when appropriate procedures are put in place, excellent concordance can be obtained between measurements conducted by AMS and LSC (Kaye et al., 1997; Garner et al., 2000; Young et al., 2001). In the present study, plasma levels of unchanged Compound A after a 1 mg/kg oral dose, measured by a specific LC-MS/MS assay, proved to be closely similar to those of total 14C assessed by AMS. Therefore, it could be concluded that Compound A accounted for virtually all of the drug-related material in the circulation of dogs given a pharmacological dose of this agent. However, for compounds that are extensively metabolized, it would be necessary to fractionate the plasma samples to obtain a more accurate quantification of parent levels. Fractionation of samples by HPLC followed by AMS analysis allows the separation of various individual components that account for total radioactivity in the samples being analyzed (Garner et al., 2002). When the animals were treated with Compound A at a microdose (0.02 mg/kg), either orally or by the intravenous route, the limited sensitivity of the LC-MS/MS assay did not allow plasma concentrations of parent drug to be followed beyond the 2-h time point, whereas levels of total 14C remained well above limits of detection by AMS through the final (80-h) time point. More importantly, the greatly enhanced sensitivity of AMS revealed the multiphasic kinetic profile of Compound A, which was not evident in the corresponding data set from the LC-MS/MS analyses, thereby reinforcing the need for AMS in microdose studies. The low levels of radioactivity used in this study precluded the use of LSC for determining total 14C levels in plasma at all dose levels.
Previous reports on the use of AMS strategies in support of early drug development have focused largely on the benefit associated with exposure of human subjects to very low levels of radiation (Young et al., 2001; Garner et al., 2002.). According to regulations in the UK, exposure to such low levels of radioactive test material does not require regulatory approval since the level of exposure to ionizing radiation from such small doses is lower than that expected from the background environment. The dose of radioactivity used in the present dog study was at least 500- to 1000-fold lower than would be used for a conventional high-dose radiotracer study in which LSC was to be used for monitoring drug-related material in plasma and excreta. In the United States, radiological materials with specific activities less than 50 nCi/g can be disposed of by licensed radioactivity users as nonradioactive waste to a limit of 1 µCi per year (as stipulated in the Code of Federal Regulations 10-CFR-20.2005).
Currently, there are two published examples of clinical AMS studies in which the disposition of pharmaceuticals in humans was defined after administration of low levels of 14C-labeled drugs (Young et al., 2001; Garner et al., 2002). The study reported in the present communication highlights a further benefit of AMS for pharmaceutical applications, namely, the definition of the full pharmacokinetic profile of a test compound, even after a microdose, that would not have been possible by conventional techniques due to limitations of sensitivity. Human pharmacokinetic measurements also have been determined over long time periods using AMS to quantify low levels of nutrients in a general population including healthy young adults (Dueker et al., 2000; Lemke et al., 2003).
In summary, the current study successfully validated the technique of accelerator mass spectrometry by investigating the disposition of Compound A in dogs, which afforded pharmacokinetic data in excellent agreement with those obtained via conventional techniques. The study also validated for the first time the use of a microdose strategy to assess the full pharmacokinetic profile of a drug candidate by demonstrating linearity across a 50-fold dose range. Investigations of this type in animal species should prove valuable in assessing the suitability of microdose/AMS approaches for exploratory pharmacokinetic studies in human subjects.
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Acknowledgments |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: AMS, accelerator mass spectrometry; AUC, area under the plasma concentrations versus time curve; Compound A, 7-deaza-2'-C-methyl-adenosine; Cmax, maximum concentration in plasma; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LSC, liquid scintillation counting; Tmax, time of occurrence of maximum concentration in plasma; Vdss, steady-state volume of distribution.
1 Current address: Department of Pharmacokinetics and Drug Metabolism, Amgen Biologicals, Thousand Oaks, CA 91320. 
Address correspondence to: Dr. Punam Sandhu, Department of Drug Metabolism, WP75A-203, Merck Research Laboratories, West Point PA 19486. E-mail: punam_sandhu{at}merck.com
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ina late Pleistocene hominid remains. Proc Natl Acad Sci USA 96: 12281-12286.This article has been cited by other articles:
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  J. S. Bertino Jr, H. E. Greenberg, and M. D. Reed American College of Clinical Pharmacology Position Statement on the Use of Microdosing in the Drug Development Process J. Clin. Pharmacol., April 1, 2007; 47(4): 418 - 422. [Full Text] [PDF]
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 S. K. Balani, N. V. Nagaraja, M. G. Qian, A. O. Costa, J. S. Daniels, H. Yang, P. R. Shimoga, J.-T. Wu, L.-S. Gan, F. W. Lee, et al. EVALUATION OF MICRODOSING TO ASSESS PHARMACOKINETIC LINEARITY IN RATS USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY Drug Metab. Dispos., March 1, 2006; 34(3): 384 - 388. [Abstract] [Full Text] [PDF]
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) and total 14C levels (ng Eq/ml; 
