Article pubs.acs.org/molecularpharmaceutics
Comparative Physiology of Mice and Rats: Radiometric Measurement of Vascular Parameters in Rodent Tissues C. Andrew Boswell,* Eduardo E. Mundo, Sheila Ulufatu, Daniela Bumbaca, Hendry S. Cahaya, Nicholas Majidy, Marjie Van Hoy, Michelle G. Schweiger, Paul J. Fielder, Saileta Prabhu, and Leslie A. Khawli*,† Genentech Research and Early Development, South San Francisco 94080, United States S Supporting Information *
ABSTRACT: A solid understanding of physiology is beneficial in optimizing drug delivery and in the development of clinically predictive models of drug disposition kinetics. Although an abundance of data exists in the literature, it is often confounded by the use of various experimental methods and a lack of consensus in values from different sources. To help address this deficiency, we sought to directly compare three important vascular parameters at the tissue level using the same experimental approach in both mice and rats. Interstitial volume, vascular volume, and blood flow were radiometrically measured in selected harvested tissues of both species by extracellular marker infusion, red blood cell labeling, and rubidium chloride bolus distribution, respectively. The latter two parameters were further compared by whole-body autoradiographic imaging. An overall good interspecies agreement was observed for interstitial volume and blood flow on a weight-normalized basis in most tissues. In contrast, the measured vascular volumes of most rat tissues were higher than for mouse. Mice and rats, the two most commonly utilized rodent species in translational drug development, should not be considered as interchangeable in terms of vascular volume per gram of tissue. This will be particularly critical in biodistribution studies of drugs, as the amount of drug in the residual blood of tissues is often not negligible, especially for biologic drugs (e.g., antibodies) having long circulation half-lives. Physiologically based models of drug pharmacokinetics and/or pharmacodynamics also rely on accurate knowledge of biological parameters in tissues. For tissue parameters with poor interspecies agreement, the significance and possible drivers are discussed. KEYWORDS: physiology, drug delivery, blood flow, interstitial volume, vascular volume, interspecies scaling
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occupied by blood and interstitial fluid. As such, preclinical methods for measurement of vascular volume (Vv) have been developed to allow subtractive correction for the amount of drug within the blood of tissues.2 In addition, similar methods have been established for measurement of extracellular volume (Ve) following intravenous infusion of an extracellular marker.1 Subtracting the vascular volume from the extracellular volume allows derivation of the pharmacologically relevant quantity, the interstitial volume (Vi), which is required for calculation of drug concentrations within the interstitial space. Numerous drugs are targeted toward receptors that reside within the interstitial space, the fluid-filled compartment that lies between the outer endothelial vessel wall and the plasma membranes of cells.9 The interstitium is also referred to as the biophase due to its central role in the biological mechanism of action for many drugs including a number of cancer therapeutic agents.10 Because
INTRODUCTION The absence of many physiological processes in vitro and interspecies differences in vivo can confound direct comparisons of in vitro, preclinical, and clinical data.1,2 A vast array of physiological data for humans and laboratory species is available in the literature;3−6 however, it should be utilized with an understanding of its limitations. Measurement techniques vary widely, and the use of assumed nominal values is common.2 Furthermore, the physiologies of disease tissues such as xenograft models are highly variable and largely unknown. Significant physiological variability across species, age, breed, disease status, drug treatment, and time of day7 motivates direct measurement of relevant physiological properties or processes whenever possible.8 Tissues may be considered to be composed of three separate physiological compartments: the intravascular, interstitial, and intracellular spaces (Figure 1). If drug concentrations are measured in terms of total, whole-tissue uptake, then a physiologically based correction is necessary to derive individual compartmental concentrations. Such corrections require knowledge of the relative tissue spaces that are © 2014 American Chemical Society
Received: Revised: Accepted: Published: 1591
December 13, 2013 March 27, 2014 April 7, 2014 April 7, 2014 dx.doi.org/10.1021/mp400748t | Mol. Pharmaceutics 2014, 11, 1591−1598
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Figure 1. Conceptual illustration of techniques used to measure physiological parameters relevant to drug uptake in tissues. The tissue is divided into intravascular, interstitial, and intracellular compartments (depicted in red, beige, and blue, respectively). The vascular volume (Vv) may be measured using 99mTc-labeled red blood cells (RBC), while the extracellular (i.e., Vv + interstitial (Vi)) space is measured by steady-state infusion of 111In− DTPA. Water molecules can freely diffuse between all three compartments. The rate of blood flow (Q) to the tissue may be measured as the proportion of a bolus dose of 86Rb+ that enters the tissue (possibly entering cells via Na+/K+ ion channels) in a brief time interval.
Inc. (Waltham, MA). Indium-111−DTPA (i.e., 111In-pentetate) and technetium-99m (99mTc) pertechnetate were purchased from Triad Isotopes (South San Francisco, CA). All three were diluted with sterile normal saline prior to intravenous dosing. TechnescanPYP (i.e., stannous pyrophosphate + 99mTc) kits for preparation of 99mTc pyrophosphate injection were purchased from Triad Isotopes and used according to label instructions for the in vivo method of blood pool imaging with the appropriate human-to-mouse or human-to-rat scaling based on relative body weight. Briefly, each reconstituted clinical kit vial of TechnescanPYP contains 4 mg of tin chloride (3 human doses) in 3 mL, while a preclinical dose of only 0.5 μg (or 5 μg for rats) of tin chloride is desired assuming a 70 kg human and 0.02 kg mouse (or a 0.2 kg rat). Animal Models. The protocol, housing, and anesthesia were approved (Protocol numbers 11-1158 and 12-0151) by the Institutional Animal Care and Use Committees of Genentech Laboratory Animal Resources, in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care regulations. Female DBA/2 mice (immunocompetent, inbred) in a 20−30 g body weight range and 6−8 weeks of age were obtained from Charles River Lab (Wilmington, MA). Precannulated (both jugular and femoral) female Sprague−Dawley rats in a 203−218 g body weight range were also obtained from Charles River Lab. In rats, ketamine
most drug targets are located in the interstitial space, interstitial concentrations are often more predictive of drug effect than total tissue concentrations. In addition to compartmental-based correction of drug concentrations in tissues, physiologically based pharmacokinetic (PBPK) modeling represents another important application for physiological parameters. PBPK models can aid in understanding mechanisms of tissue uptake and can predict, by means of interspecies scaling,11 tissue concentrations of therapeutic drugs (both small and large molecules) in humans based on preclinical pharmacokinetics data.12−14 Prediction of human pharmacokinetic parameters is often based on analogous data in rodent species. The success of PBPK models, therefore, is dependent on parameter values that accurately reflect in vivo tissue physiological conditions. Importantly, a sensitivity analysis of a previously reported PBPK model implicated Vv and Vi as two of the most influential parameters on antibody concentration in tissues, particularly at early time points.5 Consequently, we have chosen to radiometrically determine Vv, Vi, and a third critical physiological parameter, blood flow (Q),15,16 in several mouse and rat tissues.
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EXPERIMENTAL SECTION Radiopharmaceuticals. Rubidium-86 chloride (86RbCl) was purchased from PerkinElmer Life and Analytical Sciences, 1592
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and xylazine (intraperitoneal dosing) anesthetics were used for surgeries (performed at Charles River Lab), and a postoperative analgesic (buprenorphine, subcutaneous) was utilized. Rats were sacrificed by euthasol via jugular cannula. In mice, ketamine (60−80 mg/kg) and xylazine (10−15 mg/kg) (intraperitoneal dosing) anesthetics were used for surgeries (performed at Genentech), and a postoperative analgesic (meloxicam, subcutaneous) was utilized. Mice were sacrificed by cervical dislocation under inhaled isoflurane anesthesia. Vascular Volume. The intravascular spaces of rodent tissues were measured using a previously reported indirect red blood cell (RBC) labeling method.2 This measurement is based on a clinically utilized blood pool nuclear imaging protocol and relies on radiolabeling of RBCs with 99mTc (Figure 1) and measuring the amount of radioactivity in tissues and blood using a gamma counter, yielding vascular volume (Vv) in units of microliters per gram of tissue:7,8 Vv =
CPM Tc‐99m
CPM Tc‐99m
gram of tissue
microliter of blood
Vi =
CPMIn‐111 gram of tissue
CPMIn‐111 microliter of blood CPMIn‐111 microliter of plasma
−
× Vv (2)
A similar radiometal−polyaminopolycarboxylate complex, chromium-51−ethylenediaminetetraacetic acid (51Cr−EDTA), has been previously used by others in a similar context.9 As Vi is calculated from both 99mTc- and 111In-derived data, the percentages of injected doses (% ID) of 111In−DTPA were also calculated. Furthermore, because Vv values are generally smaller compared with Vi, the subtractive (blood correction) term in eq 2 does not drastically affect the calculation. In physiological terms, this suggests that the extracellular volumes (Ve) are approximately equal to interstitial volumes in tissues having lower blood content. Extracellular volume may be calculated from Vv and Vi if the volume fraction of red blood cells (e.g., hematocrit ( f)) is known: Ve = Vv(1 − f ) + Vi
(3)
Alternatively, Ve may be calculated by simply omitting the subtractive term in eq 2:
(1)
The indirect method involved transfusion of radiolabeled blood from donor rodents into study (i.e., recipient) rodents, where donor rodents had been subjected to 99mTc labeling of RBCs in vivo following administration of stannous (Sn2+) pyrophosphate.7,17 Use of the clinical Technescan PYP kit is conceptually based on the original method of Sands et al. for in situ (i.e., in vivo) RBC labeling with 99mTc.18,19 The previous administration of stannous pyrophosphate, a component of the reconstituted Technescan kit, reduces 99mTc-pertechnetate intracellularly so that it may bind to the beta chain of hemoglobin.20 Unless otherwise indicated, all agents were administered to mice via tail vein injection, while rats were purchased precannulated. Donor mice and rats received intravenous bolus doses containing 0.5 and 5 μg, respectively, of stannous pyrophosphate via a TechnescanPYP kit that was reconstituted and appropriately diluted in phosphate buffered saline. Thirty minutes later, each “pretinned” donor rodent received intravenous bolus doses of 99mTc-pertechnetate (370 MBq) in saline. For rats, the stannous pyrophosphate and 99mTcpertechnetate were administered via femoral and jugular cannula, respectively, with blood withdrawal via femoral cannula. Sixty minutes later, donor rodents were sacrificed, and the withdrawn blood (using EDTA as an anticoagulant) was pooled and carefully reinjected (0.56 MBq per rodent) into naive rodents for Vv measurement as previously described.2 At sacrifice, harvested tissues were externally rinsed with phosphate buffered saline and blotted dry prior to analysis; however, no exsanguination or perfusion of residual blood was performed, as this would prevent Vv measurement. Still, it should be noted that substantial measurement error is present due to differences in intraorgan blood storage and drainage, especially for highly perfused tissues such as the lung. Interstitial Volume. The extracellular spaces of rodent tissues were measured by continuous infusion of the extracellular marker, 111In−DTPA.21,22 Subtracting the vascular volume (using 99mTc) from the extracellular volume (111In) allows derivation of the pharmacologically relevant quantity, the interstitial volume (Vi), in units of microliters per gram of tissue.8,21
Ve =
CPMIn‐111 gram of tissue
CPMIn‐111 microliter of plasma
(4)
Precannulated rats were used to aid in dosing and blood collection. For mice, jugular cannulation surgeries were performed 48 h prior to measurement to allow recovery while maintaining a constant saline infusion at 20 μL/h. Extracellular volume measurement was achieved by administering a constant intravenous infusion of 111In−DTPA (3700 kBq/ mL) at a rate of 300 μL/h for exactly 1 h. Plasma (EDTA) and tissue samples were collected by retroorbital bleed and terminal organ harvest, respectively, and counted for radioactivity using a 1480 WIZARD Gamma Counter (Wallac, Turku, Finland) in the energy window for the 245 keV photon peak of 111In (t1/2 = 2.8 days) and with automatic background and decay correction. The interstitial (Vi) and extracellular (Ve) volumes were calculated as shown in eqs 2 and 4, respectively. Blood Flow. Regional blood flow rates (Q) in various organs and tissues were determined by sacrificing mice exactly 90 s following intravenous bolus injection of a 185 kBq quantity of 86RbCl.7,23−26 Rats and mice were dosed via jugular cannula and tail vein injection, respectively. Tissue samples were promptly collected by terminal organ harvest and counted for radioactivity using a 1480 WIZARD Gamma Counter in the energy window for the 1077 keV photon peak of 86Rb (t1/2 = 18.7 days) and with automatic background and decay correction. Blood flow (Q) was calculated as follows:7,8 ⎛ CPMRb‐86 ⎞ Q=⎜ × COtotal ⎟ ⎝ gram of tissue ⎠
CPMRb‐86 total injected dose
(5)
where total cardiac output (COtotal) = 8 and 74 mL/min for mice and rats, respectively.4 Whole-Body Autoradiographic Imaging. Mice and rats were assessed by quantitative whole-body autoradiography. Animals were processed for whole-body cryosectioning, exposed, and imaged as described previously.27,28 Carcasses were embedded in 4% carboxymethylcellulose and stored at −70 °C until sectioning. Sagittal sections of 20 μm thickness were obtained using a cryostat microtome at −20 °C. The sections were collected at levels of interest in the sagittal plane, and all major tissues, organs, and fluids were included in these 1593
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mouse but not in rat. The Vv values in μL/g for rat lung (540 ± 69), kidney (180 ± 18), and spleen (300 ± 100) were all significantly higher than for the corresponding values in mouse (160 ± 27, 110 ± 19, and 70 ± 9.1, respectively) (Table 1). The Vv values for small intestine, large intestine, muscle, skin, and stomach were also higher in rats than in mice. The thymus was the only organ tested for which the Vv was higher in mice than in rats. In liver, the Vv in rats was borderline higher than in mice. No significant differences in Vv between mice and rats were measured in heart, fat, brain, pancreas, and lymph nodes. Interstitial Volume. The Vi values of most tissues were largely similar (Figure 3). The Vi value in μL/g for rat skin (710
levels. Sections were lyophilized and mounted. The whole-body cryosections were exposed to phosphorimaging plates and scanned using a Fuji Film BAS-5000 scanner (Fuji Film Medical Systems, Inc.) to obtain digital images of the radioactivity in each section. Statistical Analysis. Data from mice and rats were compared by unpaired t test. Significant (p < 0.05) differences are indicated by asterisks.
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RESULTS Vascular Volume. The lungs, liver, kidneys, heart, and spleen represented the most highly vascularized organs in both species (Figure 2). The thymus was highly vascularized in
Figure 3. Measured interstitial volumes (Vi) of selected tissues of rats and mice. The extracellular spaces of rodent tissues were measured by continuous infusion of the extracellular marker, 111In−DTPA. Subtracting the vascular volume (using 99mTc) from the extracellular volume (111In) allows derivation of interstitial volume (Vi) in units of microliters per gram of tissue. Statistically significant differences between the two species are denoted by asterisks.
Figure 2. Measured vascular volumes (Vv) of selected tissues of rats and mice. Calculation of Vv values relied on an indirect red blood cell labeling method involving transfusion of radiolabeled blood from donor rodents into study (i.e., recipient) rodents, where donor rodents had been subjected to 99mTc labeling of RBCs in vivo following administration of stannous (Sn2+) pyrophosphate. Statistically significant differences between the two species are denoted by asterisks.
± 140) was significantly higher than for the corresponding value in mice (366 ± 78) (Table 2). Conversely, the Vi value in μL/g for rat lymph nodes (180 ± 49) was significantly lower
Table 1. Experimentally Measured Percentages of Injected Doses (% ID) of 99mTc-Labeled Red Blood Cells (RBC) with Corresponding Calculated and Literature Vascular Volumes (Vv) in Rodent Tissuesa Vv (μL/g) 99m
Tc-RBC (% ID) measured
literature3
measured
tissue
rat
mouse
rat
mouse
small intestine large intestine lungs liver kidneys heart spleenc muscle fat brain skin stomach thymus pancreas lymph nodes
0.45 ± 0.12b 0.20 ± 0.05b 6.3 ± 1.2 4.1 ± 1.9 1.9 ± 0.3 0.33 ± 0.15 0.70 ± 0.33 3.7 ± 0.3b 1.2 ± 0.1b 0.084 ± 0.015 3.1 ± 0.7b 0.12 ± 0.03 0.030 ± 0.007 0.11 ± 0.04 0.064 ± 0.040
0.55 ± 0.04b 0.17 ± 0.08b* 1.3 ± 0.2* 2.8 ± 1.1 1.9 ± 0.4 0.74 ± 0.37 0.39 ± 0.04 2.8 ± 0.3b* 1.1 ± 0.4b 0.27 ± 0.03* 1.2 ± 0.6b* 0.14 ± 0.03 0.45 ± 0.30* 0.11 ± 0.04 0.066 ± 0.034
26 ± 8 20 ± 4 540 ± 69 120 ± 75 180 ± 18 76 ± 22 300 ± 100 7.5 ± 0.7 14 ± 2 8.8 ± 1.1 13 ± 3 19 ± 4 18 ± 4 30 ± 4 25 ± 9
16.4 ± 0.4* 9.2 ± 1.7* 160 ± 27* 45 ± 13 110 ± 19* 96 ± 41 70 ± 9* 5.5 ± 1.1* 16 ± 12 10 ± 1 5.3 ± 2.6* 11 ± 2* 140 ± 60* 15 ± 14 17 ± 6
rat
mouse
260−520 120−270 110−270 260 170−280 10−90
400−620 230−360 120−340
20−40 20
30 30
170−190 30−50
a
Data from 5 animals per group; statistically significant differences between the two species are denoted by asterisks. bCalculated on the basis of literature value of mean tissue weight, as only a portion of this tissue was analyzed. cValues of Vv are likely overestimated due to the spleen’s physiological role in sequestration of damaged or aged red blood cells. 1594
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Table 2. Experimentally Measured Percentages of Injected Doses (% ID) of Interstitial (Vi) and Extracellular (Ve) Volumes in Rodent Tissuesa 111
111
In−DTPA with Corresponding Calculated
Ve (μL/g) measured
In−DTPA (% ID) measured
Vi (μL/g) measured
tissue
rat
mouse
rat
mouse
rat
mouse
small intestine large intestine lungs liver kidneysc heart spleen muscle fat braind skin stomach thymus pancreas lymph nodes
0.29 ± 0.06b 0.26 ± 0.08b 0.43 ± 0.25 1.5 ± 0.8 1.8 ± 0.4 0.12 ± 0.05 0.029 ± 0.006 4.5 ± 0.3b 0.89 ± 0.80b 0.021 ± 0.007 16 ± 2b 0.14 ± 0.02 0.04 ± 0.02 0.03 ± 0.02 0.04 ± 0.03
0.38 ± 0.27b 0.23 ± 0.15b 0.20 ± 0.07 0.40 ± 0.13* 2.1 ± 0.5 0.076 ± 0.021 0.017 ± 0.003* 3.2 ± 1.3b 3.4 ± 3.5b 0.037 ± 0.017 5.1 ± 0.9b* 0.09 ± 0.01* 0.015 ± 0.007* 0.037 ± 0.008 0.05 ± 0.02
181 ± 43 262 ± 69 529 ± 78 273 ± 83 2032 ± 393 356 ± 143 113 ± 29 99 ± 18 67 ± 25 25 ± 7 718 ± 139 253 ± 61 210 ± 53 141 ± 63 195 ± 49
229 ± 129 230 ± 87 316 ± 25* 116 ± 19* 2067 ± 474 180 ± 28 71 ± 10* 96 ± 27 462 ± 379 23 ± 3 370 ± 77* 218 ± 100 201 ± 142 103 ± 38 402 ± 178*
160 ± 43 250 ± 69 240 ± 200 240 ± 138 1900 ± 380 160 ± 130 43 ± 27 93 ± 18 57 ± 25 19 ± 7.3 710 ± 140 240 ± 60 197 ± 53 120 ± 63 180 ± 49
218 ± 130 220 ± 87 210 ± 20 76.3 ± 1.2 2000 ± 480 120 ± 25 20 ± 9 92 ± 27 460 ± 380 16 ± 3.4 366 ± 78* 210 ± 99 106 ± 150 74 ± 4.0 390 ± 180*
a Data from 5 rats and 4 mice per group; statistically significant differences between the two species are denoted by asterisks. bCalculated on the basis of literature value of mean tissue weight, as only a portion of this tissue was analyzed. cInaccurate measure of Vi due to renal filtration of 111In− DTPA. dInaccurate measure of Vi due to 111In−DTPA’s inability to cross the blood−brain barrier.
than for the corresponding value in mice (390 ± 180). No significant differences in Vi between mice and rats were measured in any other organ. The measurement of Vi in kidneys is not physiologically meaningful due to renal clearance of the small molecule radioactive probe, indium-111−DTPA, throughout the infusion. Likewise, the Vi values measured for brain are misleading due to the inability of indium-111−DTPA to cross the blood−brain barrier. Using eq 4, the Ve values in μL/g in mice for small intestine and muscle were 229 ± 129 and 96 ± 27, compared to Vi values of 218 ± 130 and 92 ± 27, respectively (Table 2). The respective values in rats were 181 ± 43 and 99 ± 18 for Ve and 160 ± 43 and 93 ± 18 for Vi. However, somewhat larger differences arise for highly perfused tissues. For example, the Ve values in μL/g in mice for liver and lungs are 116 ± 19 and 316 ± 25, compared to Vi values of 76.3 ± 1.2 and 210 ± 20, respectively. The respective values in rats were 273 ± 83 and 529 ± 78 for Ve and 187 ± 82 and 240 ± 200 for Vi. The Ve, but not Vi, in lung was significantly higher in rats than in mice. Blood Flow. The Q values of most tissues were largely similar (Figure 4). The calculated Q values in μL/g/min for rat heart (1200 ± 160), spleen (380 ± 89), stomach (370 ± 52), thymus (250 ± 45), and pancreas (500 ± 92) were significantly higher than the corresponding value in mice (830 ± 67, 220 ± 29, 160 ± 26, 160 ± 66, and 220 ± 120, respectively) (Table 3). No significant differences in Q between mice and rats were measured in any other organ. The Q values measured for brain are not physiologically meaningful due to the inability of rubidium ions to cross the blood−brain barrier. In addition to blood flow, the Q values for heart may also reflect, in part, cellular uptake of rubidium ions via active potassium ion transporters within cardiac muscle. Whole-Body Autoradiographic Imaging. The autoradiographs in Figure 5 are intended to serve as qualitative visual aids, and it is important to note that none of the data in Figures 2−4 were derived from these images. Autoradiography only captures data from the cryosections that are selected for analysis, so whole-tissue analysis by gamma counting is better suited for rapidly measuring physiology at the organ level. For
Figure 4. Measured blood flows (Q) to selected tissues of rats and mice. Regional blood flow rates (Q) in various organs and tissues were determined by sacrificing mice exactly 90 s following intravenous bolus injection of a 185 kBq quantity of rubidium-86 chloride (86RbCl). Statistically significant differences between the two species are denoted by asterisks.
both of the blood flow images in Figure 5A,C, the heart is the organ with highest uptake. The localization of radioactivity in the cardiac muscle surrounding the heart, but not within the blood itself, is apparent especially in the mouse images. High renal uptake and virtually no brain uptake are present in both species, reflecting the renal clearance of the radioactive probe and its exclusion from the blood−brain barrier, respectively. Generally low uptake was observed in the general carcass (including muscle) with particularly low uptake in gut cavities. For both of the blood volume images in Figure 5B,D, the highly perfused heart and lungs contain the highest uptake, followed by the kidneys and liver. The short decay half-life of 99mTc combined with the larger blood volume (and thus larger dilution factor) of the rat resulted in Figure 5D having the poorest image quality in terms of signal-to-noise ratio.
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DISCUSSION We previously reported the measurement of Vv, Vi, and Q in beige nude (immunodeficient) mice.1 In the current effort, we 1595
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Table 3. Experimentally Measured Percentages of Injected Doses (% ID) of 86RbCl (Rubidium-86 Chloride) with Corresponding Calculated and Literature Blood Flows (Q) in Rodent Tissuesa Q (μL/g/min) 86
RbCl (% ID) measured
tissue
rat
small intestine large intestine lungs liver kidneys heart spleen muscle fat brainc skin stomach thymus pancreas lymph nodes
4.4 ± 1.6 2.4 ± 1.0b 1.1 ± 0.2 3.2 ± 0.4 8.4 ± 1.2 1.8 ± 0.3 0.42 ± 0.12 26 ± 5b 1.7 ± 0.8b 0.059 ± 0.007 9.2 ± 0.6b 0.90 ± 0.10 0.18 ± 0.04 0.72 ± 0.13 0.099 ± 0.022 b
literature3
measured
mouse
rat
mouse
5.3 ± 0.8 1.4 ± 0.1b 0.94 ± 0.17 3.0 ± 0.4 9.8 ± 2.4 1.8 ± 0.1 0.35 ± 0.07 19 ± 4b 1.5 ± 1.1b 0.08 ± 0.04 4.1 ± 2.2b* 0.61 ± 0.10* 0.11 ± 0.04* 0.74 ± 0.18 0.23 ± 0.21
610 ± 230 550 ± 240 240 ± 39 180 ± 34 2500 ± 1000 1200 ± 160 380 ± 89 130 ± 25 48 ± 23 15 ± 1.4 95 ± 6 370 ± 52 250 ± 45 500 ± 92 150 ± 65
420 ± 280 350 ± 27 360 ± 140 170 ± 19 1500 ± 850 830 ± 67* 220 ± 29* 130 ± 29 57 ± 43 10 ± 4* 66 ± 35 160 ± 26* 160 ± 66* 220 ± 120* 250 ± 150
b
rat
mouse
380−1470 90−480 4220−8260 4050−7170
350 200 4220−4950 7680−7930
150−470 180−480 450−1340 60−220
200−280 840−850 90−260
a
Data from 5 rats and 4 mice per group; statistically significant differences between the two species are denoted by asterisks. bCalculated on the basis of literature value of mean tissue weight, as only a portion of this tissue was analyzed. cInaccurate measure of Q due to 86RbCl’s inability to cross the blood−brain barrier.
evaluation. The use of DBA/2 mice, as opposed to nude or SCID mice, allows for direct comparison to a commonly utilized immunocompetent rat model. Inclusion of additional tissues allows a more complete analysis of organ physiology. This is especially true for skin and large intestine, each of which represents a significant fraction of body weight. Overall, the agreement between mice and rats for 2 of the measured physiological parameters, Vi and Q, was excellent in most of the 15 tissues analyzed. Significant interspecies differences were observed in only 2 tissues for Vi (Figure 3), while 5 tissues were significantly different for Q (Figure 4). However, Vv was higher for rats in 8 tissues (and lower in 1) (Figure 2). These findings do not agree with literature sources which report Vv values for rats that are in similar ranges, often trending lower, as compared with mice (Table 1).3 However, these seemingly contradictory findings must be weighed against the wide range of literature values and the differences among measurement techniques. For instance, the range of literature Vv values collated by Brown and colleagues3 (see Table 1) were measured by numerous techniques including the use of radioiodinated proteins,29 chromium-51-labeled red blood cells,30 and a combined approach involving both iron-59labeled red blood cells and iodinated albumin.31 Although albumin is indeed largely excluded from the interstitium due to electrostatic interactions,32 we selected radiolabeled red blood cells for measuring blood volume due to their large size and inability to extravasate. Alternatively, the observed interspecies differences in Vv may originate from biological differences between mice and rats. For instance, the splenic architecture in rats is characterized by large venous sinuses, while murine spleens are nonsinusal.33 A greater abundance of red blood cells in the sinusal rat spleen relative to nonsinusal murine spleen may contribute to the difference in Vv that we observed (Figure 2, Table 1). Adding to this picture, it is possible that the spleens of mice and rats might process damaged or aged RBCs differently. In addition, rat lung was shown by Faffe et al. to contain a significantly greater volume proportion of blood vessel wall (and lower alveolar
Figure 5. Whole body autoradiographic imaging of mice (A, B) and rats (C, D) to illustrate the patterns of blood flow (A, C) and blood volume (B, D). Each panel (A−D) contains two nonadjacent cryosectioned sagittal slices from the same animal, one (slice 1) that is through the midlane plane (centrally through the heart) and another (slice 2) that is offset and more likely to contain kidney. Both a digital photograph (dig) and a corresponding pseudocolored autoradiograph (aut) image is included for each slice. Blood flow images depict the distribution of a blood flow tracer, rubidium-86 chloride (86RbCl), 90 s after bolus injection. Blood volume images depict the distribution of circulating red blood cells (RBC) that were labeled with 99mTc. The following tissues are labeled in each image: brain (b), heart (h), lungs (lu), liver (li), kidneys (k), and gut (g).
have built on this work in a number of manners: by use of DBA/2 (immunocompetent) mice, by including 6 additional tissues (skin, stomach, thymus, pancreas, lymph node, and large intestine), and by directly comparing to rats in a side-by-side 1596
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wall) than mice (6.2 ± 0.7 and 2.3 ± 0.6%, respectively).34 This nearly 3-fold difference in blood vessel content is similar in magnitude to the difference in Vv that we observed. In contrast to the rat, the thymus in mice lacks sublobulation (i.e., thin bands of connective tissue),35 but it is unclear how this difference would rationalize our finding of a higher Vv value in mice relative to rats. To further complicate matters, a radiolabeled microsphere study showed that hepatic and renal values for Q in rats were approximately twice the corresponding values for mice16 despite the good agreement found by us (Table 3, Figure 4) and others.3 Most of the Q values reported by Brown and colleagues were measured by radiolabeled microspheres, whereas we have utilized 86RbCl. The use of these different blood flow tracers may explain why our measured Q values are considerably lower than the literature values in Table 3. Although the majority of animal treatments and procedures were identical between mice and rats, there were a few minor differences. Surgeries for both species were performed under ketamine and xylazine, but a different postoperative analgesic was utilized. However, it is important to note that we included two full days of recovery between the days of surgery and study initiation. Furthermore, euthanization was achieved by euthasol (which acts almost instantaneously) for rats but by cervical dislocation under inhaled isoflurane (induced in 1−2 min) for mice. Although anesthetics can have important effects on physiology, we believe that the anesthetic used for mice at sacrifice had minimal effects on the parameters that we measured due to the brief duration of administration. We have found reasonably good agreement in Vv, Vi, and/or Q across several different strains of mice including beige nude,1,2 C.B-17 Icr SCID,27 C.B-17 SCID beige mice (Vv and Vi only),36 and C57BL/6 (see Tables 1−3 in the Supporting Information). For instance, the Vv values (in μL/g) across the five mouse strains that we have studied ranged from 142 ± 9 to 235 ± 111 in lung, 37 ± 7 to 55 ± 11 in liver, 77 ± 7 to 110 ± 19 in kidney, and 30 ± 7 to 96 ± 41 in heart (Table 1 in the Supporting Information), with similarly good agreement for Vi and Q (Tables 2 and 3 in the Supporting Information). However, we had never previously measured these parameters in rats until initiating the current work. To ensure that the interspecies differences in physiology were reproducible, we repeated the measurement of Vv and Q in a separate study using the same methodology and identical rat strain (see Table 4 in the Supporting Information). The overall excellent agreement between the two studies for the vast majority of parameters gives further support to the notion that the agreement between mouse and rat is better for Q than for Vv in most tissues. The results in the current report will have important implications in converting whole-tissue drug concentrations into blood-corrected interstitial concentrations. For instance, vascular and interstitial volumes may be utilized to calculate the amounts of drug in the appropriate compartments. First, the amount of drug within the blood of tissues is computed: micrograms blood = C bVv × grams of tissue
C tissue,bloodcorrected =
micrograms total − micrograms blood gram of tissue (7)
Finally, the interstitial concentration is calculated using the fractional interstitial volume (ϕ): Ci =
C tissue,bloodcorrected ϕ
(8)
The fractional interstitial volume (ϕ) may be easily derived from interstitial volume (Vi) data in traditional units of volume per gram of tissue. For example, a tissue having an interstitial volume of 100 μL of blood per gram of tissue has a fractional interstitial volume of 0.100 since each gram of tissue occupies 1,000 μL of space (assuming a tissue density of 1 g/mL, as the density of most visceral organs ranges from 1.02 to 1.06).3 The same relationship exists between the fractional vascular volume (γ) and vascular volume (Vv). Once the interstitial concentration of drug is calculated, its units may be converted from micrograms per milliliter to nanomoles per liter using the drug’s molecular weight. In conclusion, we have systematically measured interstitial volumes, vascular volumes, and blood flows in selected tissues of both mice and rats by extracellular marker infusion, red blood cell labeling, and rubidium chloride bolus distribution, respectively. Aside from its utility in compartmental-based corrections of drug concentrations, this collection of physiological data in rodents will be extremely applicable in physiologically based pharmacokinetic modeling of drug concentration and effect. The in vivo kinetic behavior of both small13,37 and large5,6,12,14,38,39 molecule drugs may be predicted in humans based on preclinical data, but the success of such models is dependent on many factors including accuracy of physiological parameters.
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ASSOCIATED CONTENT
S Supporting Information *
Comparison of blood flows and vascular and interstitial volumes in different mouse strains. Reproducibility of vascular volume and blood flow in rats. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Present Address
† University of Southern California, Keck School of Medicine, Department of Pathology, 2011 Zonal Avenue, HMR 205, Los Angeles, CA 90033.
Notes
The authors declare the following competing financial interest(s): All authors are (or were) employees of Genentech, Inc., a member of the Roche Group, and have held financial interest in F. Hoffmann-La Roche.
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ACKNOWLEDGMENTS We thank Devin Tesar, Gregory Ferl, Jan Marik, Jessica Couch, Joseph Balthasar, Kapil Gadkar, Sid Sukumaran, and Jaime Anguiano for scientific discussions and Bernadette Johnstone, Cynthia Young, Elizabeth Torres, Jason Ho, Jose Imperio, Kirsten Messick, Nicole Valle, Nina Ljumanovic, Roxanne
(6)
where Cb is the drug concentration in blood expressed in micrograms per milliliter. Second, the amount of antibody (in micrograms) within the blood of tissues is subtracted from the total tissue uptake: 1597
dx.doi.org/10.1021/mp400748t | Mol. Pharmaceutics 2014, 11, 1591−1598
Molecular Pharmaceutics
Article
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