How Serial Sampling From An Individual Animal Reduces Inter-individual Variability
J Am Assoc Lab Anim Sci. 2015 Mar; 54(2): 139–144.
Published online 2015 Mar.
Using Dried Blood Spot Sampling to Improve Data Quality and Reduce Animal Apply in Mouse Pharmacokinetic Studies
Received 2014 Feb 18; Revised 2014 Mar 20; Accepted 2014 May 15.
Abstract
Traditional pharmacokinetic assay in nonclinical studies is based on the concentration of a exam chemical compound in plasma and requires approximately 100 to 200 µL blood nerveless per time point. However, the total blood book of mice limits the number of samples that can be nerveless from an individual animal—often to a single collection per mouse—thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a thin-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to xx µL), thus supporting series claret sampling and the generation of a consummate pharmacokinetic contour from a single mouse. Here nosotros compare plasma-derived data with DBS-derived data, explain how to prefer DBS sampling to back up discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that utilize DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a unmarried report.
Abbreviations: AUC0-tlast, AUC from time 0 until the final measured fourth dimension betoken; Cmax, maximal concentration; DBS, dried blood spot sampling; LC–MS-MS, liquid chromatography–tandem mass spectrometry; T, time corresponding to Cmax
Agreement the pharmacokinetics (pharmacokinetic) of a drug or a new molecular entity is important throughout the process of drug discovery and evolution. Pharmacokinetic studies crave the collection of a series of blood samples in sufficient volumes from both nonclinical studies (in mice, rats, rabbits, dogs, and others) and clinical trials. Plasma from these samples are and then analyzed to quantify the circulating concentrations of a drug or new molecular entity, which helps elucidate the exposure profiles (time compared with concentration human relationship) and enable the computation of its pharmacokinetic properties. Collectively, this information is used to understand the disposition of the compound.
According to estimates from the Foundation for Biomedical Research and AALAS, more than 90% of all lab animals used in biomedical inquiry are mice and rats. ane,thirteen Mice are used equally the premier research model for understanding human diseases, predominantly due to their biologic similarities to humans, small size, brusque life spans, rapid reproductive rates, and relatively low maintenance costs. The increasing availability of numerous mouse models developed specifically for different illness types, ranging from diabetes to spinal cord injuries, knockout mice, humanized mice, and others, have significantly enhanced the discovery of novel handling agents.
Historically the pharmaceutical industry has used plasma samples as the specimen for the quantification of circulating drug or metabolite concentrations for pharmacokinetic assay. Bioanalytical assays used for the quantification of plasma samples oftentimes require volumes of plasma ranging between l and 100 µL, depending on the lower limit of quantification required and the sensitivity of the instrumentation. Notwithstanding, the drove of sufficient blood volumes from an private mouse to generate the required volumes of plasma can exist challenging, given that the circulating blood volume in a 25-g mouse is approximately 1.8 mL (compared with approximately sixteen mL blood in a 250-yard rat). x Current guidelines and practices limit the maximal full volume of claret that can be nerveless from a 25-g mouse to approximately 10% to 15% of its circulating blood biweekly, that is, approximately 180 to 270 µL. 10,23,22
These guidelines and physiologic limitations necessitate the use of multiple rodents—especially mice—to generate a complete pharmacokinetic profile, that is, composite or sparse sampling. Therefore, the number of mice used during a pharmacokinetic study depends on the volumes of claret nerveless and needed for analysis. In our experience, typical pharmacokinetic or toxicokinetic studies tin can require an individual mouse for each collection time point and can result in the use of 25 mice or more per dose group or treatment, totaling more than than 100 mice per study.
Current bioanalytical methods used for plasma assay predominantly are based on liquid chromatography–tandem mass spectrometry (LC–MS-MS). The sensitivity of LC–MS-MS instrumentation has improved over the years, enabling the quantification of lower concentrations. 36 In add-on, advances take been made in collecting and analyzing small volumes of plasma. Recent developments include the introduction of dried claret spot (DBS) sampling and DBS analyses to support drug discovery and development, thus enabling the quantification of the concentrations of a drug or new molecular entity from a single ten- to 20-µL book of blood. 7,26,31,33,35
Although the awarding of DBS sampling to support drug discovery was demonstrated in the early on 2000s, 5 it was non until afterward in that decade that the technique gained broader implementation. twenty,31 A majority of the applications accept been for clinical use and supporting clinical trials, 37 virtually probable due to the meaning advantages resulting from DBS sampling, including the ease of implementation (globally, across multiple sites, remote sites that may lack proper instrumentation and infrastructure to support plasma sampling and storage), use in neonatal and special populations, 32,38 and the meaning savings in shipping costs. two
Different variations of DBS sampling, xix,39 microsampling of blood stored as a liquid, 4,sixteen and microsampling of plasma 6,17,25 have been reported. However, DBS sampling seems to be the nigh practical and widely implemented to date. 9,14
The feasibility of serial sampling in mice past using DBS sampling has been reported. 7,18,26,35 The data presented here demonstrate the adoption and advantages of DBS sampling in drug discovery for routine mouse studies, its effects on written report design, and the resulting savings in animal use adjustment with the 3Rs (reduction, refinement, replacement). 3,24,27,29 In add-on, the power to generate additional data, such every bit biomarkers or pharmacodynamics markers, from the same mouse in addition to the pharmacokinetic data is highlighted. Boosted information regarding the bioanalytical applications and challenges of DBS sampling can be establish in recent reviews. fourteen,twenty
Materials and Methods
CD1 mice were purchased from Harlan Laboratories (Indianapolis, IN), and examination compounds were obtained from Lilly Research Laboratories (Indianapolis, IN). All procedures were in compliance with the Guide for the Care and Employ of Laboratory Animals. 15 All brute studies were reviewed and approved by the IACUC. Fauna studies were conducted in an AAALAC-accredited program, and veterinary care was available to ensure appropriate animal care.
Mice were housed in groups of 3 per cage in standard open-topped polycarbonate shoebox-style cages with corncob bedding that were maintained on a 12:12-h calorie-free:dark cycle at a room temperature that ranged from 68 to 79 °F (twenty.0 to 26.one °C). Enrichment was provided in the form of small cotton pads, which the mice tear autonomously and apply to sleep on. A commercial diet (Harlan Teklad 2010, Harlan Labs, Indianapolis, IN) was provided as food. Cages were inverse every 7 d.
Mouse plasma pharmacokinetic study.
Test compounds were administered equally a single x-mg/kg oral dose formulated in 1% hydroxyethlycellulose (Dow Corning, Midland, MI) (w/five), 0.25% polysorbate eighty (Sigma-Aldrich, St. Louis, MO) (5/v), and 0.05% antifoam (v/v; Dow Corning, Midland, MI) in purified water. Blood samples were taken at 8 fourth dimension points from before dosing until 24-h afterwards dosing by using ii survival bleeds followed past a terminal cardiac bleed (iii samples per mouse) in a sparse-sampling blueprint every bit depicted in Figure i. Blood was collected into an EDTA-treated vacuum phlebotomy tube and centrifuged, and the plasma nerveless and stored (and shipped) frozen. For analysis, fifty-µL aliquots of plasma from each fourth dimension point were transferred to a 96-well plate, protein precipitated with 200 µL of 1:1 methanol:acetonitrile (containing internal standard), the supernatant was diluted with one:ane methanol:water and analyzed past LC–MS-MS.
Typical sparse-sampling mouse plasma report design (due north = 3 per fourth dimension point).
Mouse DBS study.
Test compounds were administered as a single x-mg/kg oral dose formulated in 1% hydroxyethlycellulose (w/5), 0.25% polysorbate 80 (v/v), and 0.05% antifoam (5/v; (Dow Corning) in purified water. Serial blood samples were taken from each mouse until 24 h after dosing to yield a complete profile from each mouse (due north = three mice). Blood was nerveless via a tail snip directly into a 20-µL EDTA-coated capillary and immediately spotted onto a Whatman DMPK-C DBS card (GE Healthcare Bio-Sciences, Piscataway, NJ). Tail snips were performed past removing approximately 1 mm of the tail by using a scalpel. Blood flow was initiated by gentle squeezing of the tail. Generally a fresh cut was not needed for subsequent bleeds within the same solar day, and blood collections were performed via the removal of the clot. A fresh cut (approximately 1 mm) was performed for the 24-h sample. Plastic tube restrainers were used during collections. No analgesia or anesthesia was used during blood collections. A single sample and spot was collected per time indicate. The DBS cards were immune to dry for approximately 2 h at room temperature, after which the cards were placed in a zip-top handbag, stored, and shipped at ambient temperature. For analysis, a single 3-mm disc was punched from each time indicate and extracted with 100 µL 1:1 methanol:acetonitrile (containing internal standard), and the supernatant was analyzed by LC–MS-MS (after dilution with one:1 methanol:water).
LC–MS-MS assay.
An HPLC system consisting of LC-10ADVP pumps and a SCL-10A pump controller (Shimadzu, Columbia, MD) was used in combination with an autosampler (model 215, Gilson, Middleton, WI). A Betasil C18 v-µm 20 × two.1 mm Javelin HPLC column (Thermo Electron, West Palm Beach, FL) was used. Mobile-phase solvent A consisted of HPLC-class bottled h2o:i Thou NH4HCO3 (2000:ten, v/five), and solvent B consisted of methanol:1 M NH4HCO3 (2000:10, five/v). Analytes were separated by using a linear slope starting at sixty% solvent B and increasing to 90% past 0.2 min, holding until 0.35 min, ramping to 98% at 0.36 min, and holding until 0.76 min. The HPLC column was held at ambience temperature, and a flow rate of i.5 mL/min was used with an injection book of 10 µL. Mass spectrometric data were generated by using an API 4000 triple-quadrupole mass spectrometer and acquired by using Annotator software, version 1.4 (Applied BioSystems, Foster Urban center, CA). Selected reaction monitoring transitions were optimized for each compound via direct infusion and used for quantification. Acquisitions were performed at unit resolution by using positive-ion atmospheric pressure ionization at a source temperature of approximately 700 °C and an ionspray voltage of approximately 1500 Five.
Pharmacokinetic calculations.
The AUC from time 0 until the last fourth dimension point (AUC0-tlast), maximal concentration (Cmax), and the time respective to the Cmax concentration (Tmax) were calculated for each animal's plasma and DBS profiles using Watson bioanalytical LIMS (version seven.4) from Thermo Scientific (Billerica, MA).
Results
The number of mice required to generate a complete pharmacokinetic profile is adamant past the volume of blood collected at a given time signal, as shown in Figure ii. In the current written report, the volume of plasma needed for analysis (every bit much as 50 μL) and the bioanalytical assay sensitivity needed to quantify the circulating concentrations limited pharmacokinetic sampling to iii fourth dimension points per mouse. The plasma and DBS pharmacokinetic profiles after the assistants of a single oral dose of a test compound are shown in Figure 3, and the corresponding pharmacokinetic properties are summarized in Tabular array 1. The plasma data are based on sampling of 9 mice and composite pharmacokinetic analysis, whereas the DBS data stand for serial sampling of 3 mice. The blood:plasma ratio for this study, calculated according to the DBS AUC and plasma AUC, is approximately 1.ii, indicating that the chemical compound is associated with the cellular components of the blood. 34
Number of mice needed to generate a pharmacokinetic profile representing 8 time points (based on a 25-one thousand mouse).
Time compared with concentration profiles after a unmarried 10-mg/kg oral dose administered to mice. The solid circles and corresponding contour are the average from triplicate measurements. (A) Plasma concentration contour via sparse sampling (composite contour) using a full of nine mice. (B) Dried blood spot (DBS) profile via serial sampling using three mice.
Table 1.
Comparison of plasma (sparse sampling) and DBS (serial sampling) pharmacokinetic parameters after a single ten-mg/kg oral dose to mice
| Parameter | Units | Plasma | DBS |
| No. of mice | ix | 3 | |
| AUC(0-t) | ng × h / mL | 4710 | 5700 |
| AUC(Extrap) | ng × h / mL | 4760 | 5943 |
| Cmax | ng / mL | 930 | 1080 |
| T1/2 | h | 3.five | 2.four |
| AUC/Dose | ng × h / mL / mg / kg | 471 | 570 |
In an attempt to gain efficiency, nosotros standardized the discovery DBS studies such that each pharmacokinetic study consisted of six private test compounds that were individually dosed to divide groups of mice (three mice per compound, xviii mice per written report). The DBS concentration data corresponding to the 6 test compounds were used to generate exposure profiles every bit shown in Figure 4. Pharmacokinetic assay of the exposure information and the concrete-chemic properties of the 6 compounds are summarized in Table 2. From a written report pattern perspective, this novel approach translates to a 66% reduction in the number of mice per study compared with a plasma study design such every bit that depicted in Figure 2 (18 mice compared with 54 mice).
Time compared with DBS concentration profiles later a single x-mg/kg oral dose of 6 test compounds administered as a single report. Each chemical compound was dosed individually to three mice (series sampling), and the entire study used xviii mice. DBS concentrations for all six compounds were generated in a single LC–MS/MS assay.
Table ii.
Pharmacokinetic data generated from DBS sampling and concrete–chemical data corresponding to 6 test compounds dosed individually (north = 3 mice per compound)
| Chemical compound | Molecular weight | cLogP | Aqueous solubility (µM) at pH 7.four | AUC(Extrap) (ng × h / mL) | Cmax (ng/mL) | Tmax (h) | T 1/ii |
| i | 545 | 3.02 | 29 | 15025 | 1478 | iii | v.4 |
| ii | 550 | 2.05 | 21 | 17925 | 2109 | 1.5 | iv.viii |
| 3 | 405 | 2.88 | >100 | 702 | 827 | 0.4 | 0.4 |
| 4 | 481 | 4.44 | 11 | 8218 | 775 | 1 | 12.9 |
| 5 | 449 | two.92 | >100 | 126 | 144 | 0.4 | 0.6 |
| 6 | 458 | 4.22 | >100 | 293 | 27 | 0.75 | 9.i |
In the current study, DBS sampling was also shown to allow measurement of both pharmacokinetics and pharmacodynamics data from the same mouse, resulting in less variability and more accurate depiction of the pharmacokinetic–pharmacodynamic relationship (Effigy 5). In this case, DBS sampling immune the collection of sufficient blood for both a complete pharmacokinetic profile (that is, time points at 0.v, 1, 2, iv, 8, 24 h) of the chemical compound and an additional five μL of blood for the measurement of blood glucose concentration, a pharmacodymanic marking for this compound, at each time point.
Time compared with DBS concentration profile after a single oral dose of a test compound administered to iv mice via oral gavage (black circles). Mean blood glucose concentrations measured every bit a pharmacodynamic marker from the same 4 mice at the aforementioned time points are plotted confronting the correct axis (open circles).
Discussion
The analytical sensitivity (lower limit of quantification) for a given analyte is a function of the book of plasma available for analysis and the limitations of the analytical instrumentation. Over the past decade, advances in pharmaceutical enquiry have resulted in the generation of novel compounds that are very potent and frequently tested at lower doses, resulting in smaller circulating concentrations. In parallel, major improvements in LC–MS-MS instrumentation have enabled the ability to quantify the desired concentration ranges by using smaller volumes of blood (approximately 100 µL blood drawn to get at least 25-µL plasma for assay), resulting in the power to obtain three or four blood samples from a single mouse. Historically, big claret volumes (greater than 200 µL) often were required, thus limiting the adequate number of collections from a mouse to a single terminal bleed or a single survival bleed followed by a terminal bleed (depending on the historic period and weight of the animal and the corresponding total volume of claret). In contrast, DBS sampling uses approximately 20 µL of claret per time point and avoids the need to generate plasma, thereby enabling the collection of an entire pharmacokinetic contour from a single mouse.
The information and examples nosotros present here demonstrate the utility and advantages of DBS sampling in mouse studies. DBS sampling has been and continues to be used to back up numerous analyses, ranging from newborn screening to therapeutic drug monitoring and HIV detection and screening. 8,eleven,21,30 However, its implementation across the pharmaceutical industry has been limited, probable due to the high degree of regulation, conservatism within the industry, and the lack of bioanalytical methods or advantages for changing from plasma to blood.
Plasma has become the 'gold standard' for pharmacokinetic analysis, driven by the fact that the analytical techniques available previously (predominantly LC–UV) required a 'clean' extract for analysis, such that plasma was selected as the matrix (given that whole claret was considered circuitous or 'dingy,' difficult to aliquot or pipette, and difficult to extract). Plasma information and blood or DBS data are equally valid from a PK perspective, but blood or DBS data should non be considered every bit equivalent to plasma information since that relationship will exist dependent on the blood:plasma partitioning ratio. Therefore, care should be taken when comparing DBS data with plasma data. Some pharmacokinetic parameters (that is, Cmax, AUC) are expected to differ between the plasma and DBS information, whereas other parameters (that is, Tmax, T1/2) are expected to be similar, depending on extent of partitioning and association of the individual compound with blood cells.
A fundamental concern with regard to DBS data has been the 'hematocrit effect,' which refers to a bias in the concentrations that arises due to a deviation between the hematocrit value of the sample and the standard bend used for quantification. This phenomenon could be significant in clinical trials, which might show marked inter-private variability in the study population due to different disease states, age, and fifty-fifty different ethnic backgrounds. This concern is minimized in nonclinical studies using purpose-bred animals, for which inter-animate being variability is expected to exist depression relative to clinical populations.
Implementing serial haemorrhage to generate a complete pharmacokinetic contour from a unmarried mouse generates pregnant savings in brute apply and is consistent with 3Rs principles in achieving a direct reduction in number of animals used (reduce); less restraint-associated stress due to the drove of smaller volumes of claret, improved data quality, and less variability (refine); and the collection of multiple endpoints from a single study such equally pharmacokinetic and pharmacodynamic data from same mouse (supersede). In improver, series claret sampling enables direct comparison of interanimal variability, which is not possible with composite sampling. The need to dose three mice compared with 8 or 24 mice (depending on the number of blood samples collected from each mouse, see Tabular array 2) also results in a significant savings in the amount of chemical compound needed for dosing, especially during early discovery, when availability can be very limited. Furthermore using markedly fewer mice per report translates to the use of fewer fauna cages and less husbandry overall.
Because of the depression sample volumes needed, DBS study animals may provide blood in excess of that needed for pharmacokinetics analyses; this excess sample tin can be used for the analysis of metabolites or biomarkers. A shortcoming of mouse DBS sampling, as we take used and described it here, is the fact that only a unmarried xx-µL spot is nerveless per time point; this practise may limit whatsoever subsequent reanalysis for incurred sample reanalysis (required for Good Laboratory Practices studies), or evaluation of metabolites, then forth. Compared with mice for plasma sampling (which typically are euthanized during blood sampling), those used in DBS studies usually are not euthanized and can exist reused, if desired, after an appropriate washout period. The low blood volumes needed for DBS samples allow intravenous–oral crossover studies to be executed in a single mouse (rather than involving multiple animals; data non shown). Intendance should be taken to employ alternative sites for DBS sampling (saphenous vein, mandibular vein) when intravenous doses are administered through the tail vein. An alternative study design for intravenous–oral crossover studies is to use cannulated mice (typically using jugular vein cannulas) for intravenous dosing followed by tail-snip DBS sampling. This intravenous phase can and so be followed with oral dosing and tail snip sampling—later an advisable washout period—thus generating pharmacokinetic parameters for both routes and oral bioavailability information from the aforementioned mouse.
The standardization of mouse discovery studies to evaluate a series of compounds (6 compounds in this written report) within a single report (individually dosed) enables rapid data generation because all of the samples tin exist analyzed within a single bioanalytical run (using a cassette standard bend) followed by the issue of a single final report. Overall, implementing DBS sampling to support mouse pharmacokinetic studies can non only save several thousand mice but also, when combined with the ease and simplicity of DBS sampling (given the lack of a need for centrifugation and separation of plasma), reduce the number of technical and husbandry personnel needed per report, thus reducing written report costs. Typically, teams engaged in the early discovery phases of drug development would be screening hundreds of molecules spanning diverse chemical structures, or 'scaffolds.' The ability to routinely execute similar report designs by using DBS and generate pharmacokinetic data for discovery compounds enhances the ability to correlate pharmacokinetic data (AUC, Cmax, Tmax, Ti/2, and and so forth) with physical–chemic data, helps to identify and streamline drug candidates, and provides critical scaffold-related information for pb generation and target optimization.
In improver, large numbers of mouse studies are conducted to appraise in vivo pharmacology where pharmacokinetic data are also needed. In many of these instances, special mouse models are used, including knockout mice, humanized mouse models, and athymic mice for oncology studies. These data indicate that prudent planning and well-designed studies can enhance the study outcomes, as demonstrated in Figure v.
Additional considerations that should be weighed prior to using DBS sampling for a chemical compound inbound drug development include an understanding of its unbound fraction in plasma and claret, its claret cell affinity, and the predictable hematocrit range. 12,28 Finally, several bioanalytical limitations and challenges related to DBS analysis, which are across the scope of the current article, need to be considered preemptively. 14
Acknowledgments
We give thanks Drs Anthony Borel and Sandaruwan Geeganage for sharing experimental data.
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