Quantitative Distribution Studies in Animals: Cross-Validation of Radioluminography versus Liquid-Scintillation Measurement

doi:10.1006/rtph.2000.1384

Regulatory Toxicology and Pharmacology

Volume 31, Issue 2, April 2000, Pages S33-S43

https://doi.org/10.1006/rtph.2000.1384 Get rights and content

Abstract

The results of a cross-validation of the radioluminography (RLG) and liquid scintillation counting (LSC) methods are presented. The methods for the determination of radioactivity concentrations were compared in 16 organs, after administration of ¹⁴C-labeled substances to rats. LSC measurements of two kinds were used as reference methods for RLG: (1) quantitative determination of radioactivity after conventional dissection (interindividual comparison) and (2) quantitative determination of radioactivity in tissue punches taken from the whole-body sections after they had undergone RLG measurement (intraindividual comparison). Blood standards containing known concentrations were used for calibration. For statistical evaluation log-linear regression analysis of paired concentration values and organ-specific 95% confidence intervals of the log-transformed RLG/LSC concentration quotients were compared. For most organs, the slopes of the regression lines and the means of the concentration quotients were within the defined equivalence range of 0.80–1.25. Deviations were distinctly smaller in the intraindividual comparison. For some organs, however, it became clear that found concentrations were affected by self-absorption (RLG) and by differences in sample preparation (LSC). In conclusion, quantification with RLG is a reliable and reproducible method with comparable measurement precision and greater accuracy in respect of tissue localization, compared to LSC (dissection).

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Role of efflux transporters in the absorption, distribution and elimination in rodents of a novel PDE4 inhibitor, CHF6001
2018, European Journal of Pharmaceutical Sciences
Citation Excerpt :
The formulated CHF6001 was intravenously administered to mice as bolus, via the tail vein, at a volume of 20 μL per mouse (0.6 mg/kg). Tissue distribution studies in rats were performed using the Quantitative Whole-Body Autoradiography, QWBA (FDA Draft Guidance, 2012; European Medicines Agency, n.d.; Ullberg, 1954; Steinke et al., 2000). Groups of six males and six female albino rats received a single or a repeated intravenous dose administered daily for 14 days.
CHF6001 is a new and potent PDE4 inhibitor for the treatment of human lung diseases, designed for topical administration by inhalation. In preclinical assessment CHF6001 appeared safe and devoid of emetic effect, which is typical side effect of PDE4 inhibitors in humans.
CHF6001 absorption, distribution and excretion were evaluated in rats by PO and IV administration of [14C]CHF6001; additionally the role of transporters was investigated by using transfected cells expressing either human transporters or MDR1 and BCRP KO mice.
[14C]CHF6001 intravenously administered as bolus distributed in all the tissues (with very low levels in brain and fetus) and it was mainly eliminated in bile. Following oral administration [14C]CHF6001 about half of the dose was absorbed through the gut.
In vitro, CHF6001 was a substrate of human membrane transporters MDR1 and BCRP.
In wild and BCRP KO mice CHF6001 was not detectable in brain, whereas it was measurable in Mdr1a/b KO mice.
Therefore, in animal species Mdr1a/b plays a significant role in CHF6001 disposition, limiting its distribution into brain and contributing to the safety profile observed in preclinical evaluation.
This behavior was confirmed by the results of the first human studies, where CHF6001 was safe and with no emetic effect at all the evaluated doses.
The distribution pattern of intravenous [<sup>14</sup>C] artesunate in rat tissues by quantitative whole-body autoradiography and tissue dissection techniques
2008, Journal of Pharmaceutical and Biomedical Analysis
Quantitative whole-body autoradiography (QWBA) and liquid scintillation counting (LSC) have been conducted to determine the metabolic profiles and tissue distribution of [¹⁴C] labeled artesunate (AS) injection in rats. The QWBA technique showed more accurate results in the quantification of radioactivity in 40 organs and tissues, compared to 19 organs with the LSC technique. The benefit of QWBA was especially apparent on measurements of bile, bone marrow, and gland organs; however, the LSC method produced more relevant findings than QWBA. Particularly, the LSC method allowed access to the following distribution patterns that were unavailable via QWBA performance: such as pharmacokinetic evaluation of radiolabeled AS in blood and plasma, tissue/plasma partition coefficients, conversion pathway of AS to dihydroartemisinin (DHA, an active and major metabolite of AS), unchanged AS and DHA in plasma, mass balance assessment, urinary and faecal eliminations, drug pathway with conjugation, [¹⁴C] AS binding with RBC and plasma protein, and metabolites identification. Even though the each method has its own advantages, common profiles were obtained from the two processes as shown in the results of the biliary metabolism, long-lasting metabolites, tissue distribution profiles, and multiple concentration peaks, which indicate a [¹⁴C] AS enterohepatic circulation.
In vitro and ex vivo storage phosphor imaging of short-living radioisotopes
2008, Journal of Neuroscience Methods
Storage phosphor imaging may be of value for biodistribution studies of short-living radiotracers in small animals. Efficiency, sensitivity and resolution of imaging plates for short-living radioisotopes vary considerably but linear response to many radioisotopes was shown previously. However, these properties have not been compared directly for larger series of short-living radioisotopes, and only few studies have directly compared data obtained from phosphor images of tissue slices with results from dissection biodistribution studies. Therefore, we evaluated the properties of imaging plates for 11 short-living radioisotopes (¹⁸F, ³²P, ⁶⁷Ga, ⁸⁹Sr, ^99mTc, ⁹⁰Y, ¹¹¹In, ¹²³I, ¹²⁵I, ¹³¹I and ²⁰¹Tl). We also evaluated the biodistribution of [¹²³I]FP-CIT in rat brain using both the phosphor technique and conventional dissection methods.
The imaging system showed a linear response for all tested radioisotopes over a wide range of radioactive concentrations and the efficiency, sensitivity and resolution varied greatly for the tested radioisotopes. Shielding experiments revealed the contribution of the various emission products of radioisotopes to these properties. However, quantitative biodistribution studies with radiotracers that are labeled with all tested radioisotopes, even ¹²³I, are feasible. The results from the ex vivo biodistribution study, using [¹²³I]FP-CIT as a radiotracer were similar for the phosphor imaging technique as compared to the dissection technique.
Advantages of phosphor imaging in radiotracer distribution studies in rat brain as compared to dissection experiments may be more precise measurements, possibility to reanalyze imaging data and 3D-reconstruction. In conclusion, phosphor imaging is an attractive alternative for biodistribution studies of short-living radiotracers in small animals.
Drug localization and targeting with receptor microscopic autoradiography
2005, Journal of Pharmacological and Toxicological Methods
This review is an argument in favor of better drug target identification. It presents the many merits and feasibilities of drug localization and target identification through the use of a suitable technique: receptor microautoradiography. Studies of drug targets and target bioavailability require methods with high resolution and sensitivity to gain information for understanding mechanisms of action, sound modeling, prediction of effects, and toxicity. For in vivo localization of drugs in tissues and cells, receptor microautoradiography was specifically designed to preserve both tissue structure and deposition of noncovalently bound diffusible compounds and to enable microscopic viewing, quantitative analysis, and characterization of target sites. This method and its applications are explained here. Pictorial and quantitative data are provided together with a discussion of identified targets that document the utility of receptor microautoradiography. For example, when applied to quantitative studies of vitamin D compounds, pharmacokinetic data of blood differed from those of target tissues and even among target tissues. Many of the target tissues discovered and characterized with receptor microautoradiography remained unrecognized with common ADME procedures, radioassay–HPLC, and whole-body autoradiography. For a visual overview of the multiple vitamin D targets, a drug homunculus has been composed. Such a drug or target homunculus may be created for any drug, dose, and time to aid in documenting and fingerprinting. Receptor microautoradiography also is a sensitive method. It can be used for the study of low-dose stimulatory actions of toxic substances to show relationships of receptor binding to dose-dependent reversal of effects, known as hormesis. In addition, a combination of autoradiography and immunocytochemistry with radiolabeled drug and antibodies to receptor or other cellular product permits further target characterization. In its own league, receptor microautoradiography provides unique information. Through greater detail and certainty, it can validate and complement less-sensitive approaches, decrease the failure rates of current ADMET predictions, and serve as a diagnostic tool and guide for biochemical, functional, and clinical follow-up in drug research and development.
Study on the practical use of quantitative whole-body auto- radioluminography
2004, Experimental and Molecular Pathology
The practical use of quantitative radioluminography (RLG) using reading system BAS 2000 (Fuji Photo Film Ltd., Tokyo, Japan) and STORM 820 (Molecular Dynamics, USA) was examined using a chemical matrix as an internal standard, which offers the benefits of being preservative, inexpensive, and easy to handle. The results were as follows:
- 1.
  As the water content is within the range of 65–80% for most organs and tissues, we selected a chemical matrix, Tissue-TEK containing 85% water, as a base for the preparation of a standard curve.
- 2.
  The calibration curve prepared using Tissue-Tek as the internal standard was compared with the calibration curve using liver paste preparations as the internal standard. The results showed good linearity in both cases, with almost no difference in the slopes of the two calibration curves.
- 3.
  The PSL-BG or MD counts/pixel-BG values of the lowest radioactivity concentration measured for small areas of the region of interest (ROI) showed large fluctuations with both BAS 2000 and STORM 820, but the fluctuation became less than 15% at above 25 mm² of ROI.
- 4.
  The value of dpm/g calculated using the calibration curve prepared from Tissue-Tex internal standards showed a very good correlation with the values of dpm/g obtained by scraping off the tissue from the remaining block and conducting measurements with a liquid scintillation counter.
Altered drug disposition of the platelet activating factor antagonist apafant in mdr1a knockout mice
2002, European Journal of Pharmaceutical Sciences
The aim of the present study was to determine a potential impact of P-glycoprotein (P-gp) on the tissue distribution and disposition of apafant (WEB 2086, CAS 105219-56-5), a selective platelet-activating factor antagonist, and on digoxin in mdr1a(−/−) and wildtype mice. Transport experiments in Caco-2 monolayers at low concentrations (<10 μM) showed that the secretory flux of [¹⁴C]apafant and [³H]digoxin exceeded the absorptive flux nine times. This efflux was concentration dependent and subject to inhibition by the P-gp substrates verapamil and cyclosporin A. This indicates that active drug transporter P-gp was involved in apafant and digoxin absorption. Mdr1a(−/−) mice showed a more than 70-fold higher concentration of digoxin-related radioactivity (P<0.001) in the brain than wildtype mice after intravenous doses of 0.05 mg/kg [³H]digoxin. Differences were less pronounced in other tissues. Both liquid scintillation counting and whole body autoradiography yielded comparable results and they also matched recently published data. Apafant-related radioactivity was about ten-fold higher in the brain of mdr1a(−/−) mice compared to wildtype mice following intravenous doses of 2 mg/kg radiolabelled apafant. Only slight or negligible differences were observed in other tissues. In wildtype mice, intestinal excretion of [¹⁴C]apafant (54.9%) exceeded biliary excretion (26.5%). However, in mdr1a(−/−) mice biliary excretion (50.7%) exceeded intestinal excretion (6.8%). These differences were mirrored in the urinary and faecal excretion. Pharmacokinetic parameters of apafant and radioactivity did not differ between wildtype and mdr1a(−/−) mice. The conclusions were: (1) apafant and digoxin are P-gp substrates, and (2) absence of mdr1a encoded P-gp significantly alters tissue distribution (especially in brain) and excretion routes (biliary and intestinal) of apafant.

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Regular ArticleQuantitative Distribution Studies in Animals: Cross-Validation of Radioluminography versus Liquid-Scintillation Measurement

Abstract

Regul. Toxicol. Pharmacol.

Regul. Toxicol. Pharmacol.

Regul. Toxicol. Pharmacol.

Regul. Toxicol. Pharmacol.

Regul. Toxicol. Pharmacol.

Imaging plate: Validation and first experience with quantitative studies in whole-body autoradiography during drug development

Xenobiot. Metab. Dispos.

Whole-Body Autoradiography

Autoradiography of new era replacing traditional X-ray film

Cell Technol.

Regular Article
Quantitative Distribution Studies in Animals: Cross-Validation of Radioluminography versus Liquid-Scintillation Measurement