Tissue Distribution Studies by Quantitative Whole Body Autoradiography

Shin-Ichiro Nagatsuka ADME & Tox. Research Institute Sekisui Medical, Co., Ltd. Sep28, 2011

QWBA webinar

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ADME studies using radioisotopes Whole body section preparation Phosphor imaging system Validation of QWBA (15 facilities) Applications

Early Research
Lead Optimization

Preclinical

Clinical Trials

FDA Review

Market (PMS)

Stability

GMP Synthesis

RI Synthesis Pharmacology

GMP RI Synthesis

Material

In vivo ADME In vitro DMPK

General Toxicology Specific Toxicology

Humanized Animal MD
Hot ADME Clinical Trial

Animal Human

Bioanalysis
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Biomarker

Radioisotopes used in QWBA all available in radiolabeling service

max beta ray energy 156 keV Specific radioactivity ca.60 mCi/mmol Molecular skeleton labeling, metabolically stable max beta ray energy 18.6 keV Specific radioactivity ca.25 Ci/mmol One step synthesis, high specific radioactivity main IC electron energy Specific radioactivity Usually used for labeling of HMW drugs
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14C

3H

125I

35 keV ca.2200 Ci/mmol

Why XenoTech-Sekisui for radiolabeling service?

45-year history and experienced radiochemists 14C >3800 compounds since 1989 3H >1000 compounds since 2000 Consultation on design of metabolically stable position and synthetic route appropriate for the target studies. Seamless offer from radio-labeling services to following in vitro / in vivo services at one site Storage and re-purification of radio-labeled compounds for repeated and future studies
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ADME studies using radioisotopes

Intestinal absorption animals humans First pass metabolism animals humans Liquid Scintillation Counting dissected tissues Whole body autoradiography various tissues in sections

Qualitative analysis metabolite ID Quantitative analysis authentic samples

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Mass Balance urine, feces, expired air, carcass Whole body autoradiography residual and/or accumulation

QWBA webinar

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ADME studies using radioisotopes Whole body section preparation Phosphor imaging system Validation of QWBA (15 facilities) Applications

Radiolabeled drug administration

Ether Halothane, Isoflurane Carbon dioxide

Euthanasia

Shaving

Frozen in dry ice-acetone

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Rubbing CMC paste on frozen body

Put into CMC paste and frozen

Cut into thin sections 30m 6 positions

Dried in the microtome

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3 Sections cover most of major tissues in rats

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Factors affecting the quality of tissue sections

Sharpness of the knife Stability of animal body in the block Pressure control when cutting

Poor

Fine

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Cracks

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QWBA webinar

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ADME studies using radioisotopes Whole body section preparation Phosphor imaging system Validation of QWBA (15 facilities) Applications

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Internal Standard

Internal Standard

External Standard nCi/g 163 0.726

476 BG

13.2

762

22.6

946

42.8

1390

103

(PSL-BG) /A = PSL/mm2 BG PSL/mm2

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Photostimulated luminescence (PSL) intensity

Y=aX+b W=1/Y

Log Y = a Log X + b Y = bXa

Radioactivity (nCi/g)
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Phosphor imaging plate system

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Highly sensitive, linear and wide dynamic range High resolution (25-100 m/pixel) Trapped energy is released by VL or Repeatedly usable after erasing not for plates for tritium

QWBA webinar

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ADME studies using radioisotopes Whole body section preparation Phosphor imaging system Validation of QWBA (15 facilities) Applications

Uniformity of 2D phosphor imaging plate layer

[14C]radioluminography standard Polymethylmethacrylate 40cm x 20cm x 2mm thick Raytest
Line1 Line 32 Row 16

9/15

Row 1

Row 1-16 : Line 1-32 : 512 ROI :

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100 1.5 % 100 1.5 % 100 2.5 %

5/15

1/15

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Reproducibility (slope of log-log linear regression)

Day1 IP1 IP2 IP3 IP4 IP5 IP6
22 Inter-plate 2.25% Min 0.51% Max 5.50% Intra-plate 2.40% Min 0.69% Max 7.73% Inter-day Min Max 1.96% 0.46% 3.73%

Day2

Day3

Day4

Day5

Day6

Variation of PSL within 15 facilities

Max Average Min

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Radioactivity (kBq/g)

External Standard
Calibrated plastic standard

Internal Standard
Blood with different RI conc.

Stable, Easy Calibration is required Section thickness must be identical

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No need to calibrate Applicable to different section thickness Time-consuming

Validation of section thickness

Intra-section CV Inter-section CV Inter-block CV

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2.45% 2.93% 3.86%

Validation Items
Scanner

Surface response uniformity Intensity against same RA source

Tissue section

Section quality Section thickness (External standard) Standard curve quality (Internal standard)
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Comparison with LSC for dissected tissue

[QWBA] = 0.944 x [LSC] r2 = 0.893 p<0.001 LSC (kBq/g)

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QWBA vs LSC High sensitivity for small tissues (1-2 dpm/mg) Possible to analyze small (part of ) tissues Possible to analyze unexpected distribution Less contamination

Self absorption (Fat, Bone) Technical expertise, cost, time

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QWBA webinar

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ADME studies using radioisotopes Whole body section preparation Phosphor imaging system Validation of QWBA (15 facilities) Applications

Semi-quantitative WBARG

Measured Calculated (PSL-BG)/A dpm/g Blood Cerebrum Liver Kidney medulla 82.61 1.03 91.27 151.45 29.8 2640.2 4381.1

Radioactivity dpm/g 2389.7

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Pregnant animals
17th day gestation 19th day gestation

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Pregnant monkey

Fetal/neonatal micro distribution Residual analysis in large animals

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Fetal mouse

Mass Spectrometry Imaging (MSI)

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Proteome analyses Skimming General analyses Chemical spotting Protease digestion Matrix MALDI-TOF imaging 200m Air brush spray Matrix IT-TOF imaging 10m

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Shimadzu AXIMA Performance (MALDI-TOF MS imaging)

Shimadzu Mass Microscope (IT-TOF MS imaging)

Chemical printer
trypsin

Shimadzu CHIP-1000
Trypsin (0.1 mg/mL 5~100 mM ABC)
5 drops x 15-30 cycles (total 7.5-15 nL)
DHB

DHB for matrix

(25 mg/mL MeOH/0.1~0.5%TFAaq.(1:1)) 3 drops x 25-33 cycles (total 7.5-10 nL) (~100 pL/ drop) 35

MALDI-TOF MS images

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IT-TOF MS images

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Example of MS-MS analysis

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QWBA in Xenotech-Sekisui
Various animal models with various dosing routes

High quality tissue section preparation

Validated phosphor imaging plate system

Related services available (Ex vivo ARG, micro ARG, ADME, RI) MS imaging applications are currently under development
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Thank you for your attendance!

Shin-Ichiro Nagatsuka XT project R&D ADME & Tox. Research Institute Sekisui Medical Co., Ltd. nagatsuka@admet.sekisuimedical.jp www.sekisuimedical.jp

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Questions and Answers

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Transcript for Q&A Session of Tissue Distribution Studies by Quantitative Whole Body Autoradiography
Could you describe a typical QWBA study the number of animals, the time to complete, typically what you perform at Sekisui? In the typical case, 4-5 time points to obtain the tissue distribution. 1 time point usually means 1 animal. That means that 4-5 animals are needed. That is the usual case. Is there a difference in the results between albino rats versus pigmented rats? This is particularly in terms of melanine binding. How is it determined in QWBA? We use Long-Evans rat for the pigmented animal study. In the case of Long-Evans rat, we can detect binding of radioactivity in melanine in the eye which is seen in chloroquine or other antibiotics. So, in order to evaluate such material in the eyes we need to use a pigmented animals. What kind of data can the client get in the final report? What kinds of units are used for the results, like nanocuries per gram of tissue, etc.? First, nanocuries per gram of tissue are obtained, but after that we use specific radioactivity of test compound to convert the results into microgram equivalent of drug gram tissue. We offer the table of the converted concentrations of the data obtained. We of course offer the picture or image of the animals. Shin, you mentioned something about sensitivity to temperature regarding exposure of plates. Could you explain that in a little bit more detail? The source of the photostimulated luminescence is trapped electrons in a hole in the higher level of excitation. That temperature gradually releases the trapped electrons. Actually, when the contact exposure is performed in a cool condition, such as -20*, 80* normally the intensity of PSL becomes higher (because electrons in a hole are preserved). Losing the trapped energy by temperature is very, very slow process. But, I recommend to perform the reading of an imaging plate right after contact exposure, but if you leave the imaging plate at no more than room temperature for more than 24 hours the reduction of PSL is usually only 5% or so. So, the temperature dependent fading is not so rapid, its a gradual process. Could you mention who the provider of the plastic standards is? For the standard to check the uniformity of response we use the plastic standard obtained from Raytest, a German company. For the standard for the quantitation we use a plastic standard obtained fromAmersham (currently Quotient) How many QWBA studies are performed at your facility every year? Are human dosimetry calculations performed? In the case of rat, we prepare 100-200 in one year. That is the most frequently used animal in QWBA. In the case of mouse its 20-40 in one year. In the case of monkey,

its less than ten. We also perform several Long-Evans rat studies for melanine binding and this is contained within the whole number of rat studies (100-200 per year). Yes, we often do human dosimetry. In the case of microdosing study in Japan, we need to estimate the possible absorption dose. Its not required in Europe, I think. If youre performing microdosing studies you dont have to perform the dosimetry, but in Japan it is required. Have you had any success in the extraction of frozen tissue sections from the QWBA for metabolite analysis? Yeah, weve tried several times for such experiment, but you know, after the preparation of tissue sections, from the remaining frozen part we can take out the part of the tissues, but such studies are restricted for the larger tissues, such as liver and so on. What is the current progress of IT TOF imaging is. Is it validated for client use, and can we get cold and radio-labeled compound information from micro distribution in specific tissues? Actually, recently there were several reports concerning IT TOF imaging to detect the chemical structure of metabolites and peptide fragments We are now trying to do that, and actually, there are several papers appearing at present. And I hope that it can be done, but there are several difficult problems different quality of each tissue and so on. So, its really hard to perform the quantitative analysis. Its restricted to semi-quantitative or qualitative analysis. There is some interest to know on experience in using double labeled tritium or carbon-14 molecules in QWBA. Actually, I have experienced double tracer autoradiography, by coupling iodine-123 and carbon-14. In the case of tritium and carbon-14 its a little bit difficult because both half-lives are long, so in order to separate the effects of both radionuclides, maybe you should use some kind of foil. If autoradiography is performed without being covered with foil, the image should be the sum of the tritium and carbon-14. If you set the appropriate foil, the weak beta radiation from the tritium is cut and you can only obtain the carbon-14 radioactivity image, but the subtraction is a little bit difficult because it is dependent on the difference of the foil thickness and so on. Ive already experienced the very simple method of using the difference half-life, and in that case, I used iodine-123 which has a very short half-life and carbon -14. The technique of QWBA has been a standard for a number of years, so what advances do you foresee in the future? You mean advances for the standard? Yes, I think it is the advances of QWBA in general. I know theres new technology coming out.

Actually, yeah, there are several new technologies, but I believe that in order to perform the microanalysis for the very small tissues, QWBA is really, really important. I think its a little bit hard to try to find out another method to perform the microanalysis of the fetal tissues. In order to determine radioactivity concentration, there might be some other method. Actually, scintillation counting is a very good method for large tissue. I was misunderstanding the question. The answer might be replaced by the following: Liquid scintillation counting is good for analysis with large volume. And QWBA is good for microanalysis for small tissue. High resolution required for microanalyses usually reduce the sensitivity, but there are many efforts to increase both sensitivity and resolution in the field of autoradiography. Could you talk a little bit about the experience that we have with micro autoradiography? Usually, in most cases, we perform the micro autoradiography for dermal application (of drugs), and in the case of micro autoradiography we normally take sections onto the slide glass, normally we use the cover glass because slide glass contains much potassium. It involves high natural radiation, so it disturbs the micro autoradiography. So, the section is taken onto cover glass (which is thin and includes less potassium). Then the photosensitive emulsion covers the section and we will count the grain (formed in the emulsion). Such method is hard to perform the quantitative analysis. We often perform the semi-quantitative analysis or the qualitative analysis of the radioactivity. When the dermal absorption is insufficient, we can judge what layer of the skin prevents the absorption of the drug. We have a lot of questions about technique here. Have you utilized the tape transfer process for QWBA? Weve tried to transfer the sections to a glass surface or something like that, but its actually really difficult. In order to stain the original sections weve once tried to transfer the sections onto the glass surface. In order to stain the sections, normally scotch tape can be used for the direct staining, and weve also tried that. I think the transfer is a little bit difficult and it needs very high technical expertise. Back to the autoradiography do you match ARG images to the tissue location on the sections? That can be done and normally, if the system accepts the DICOM images (DICOM is a name of format for image storing), its very easy to perform the superimposition of images. So, maybe we can take a tissue section image by using a video camera and store the image using the DICOM format. In the case of imaging plate system, its a very basic format and that should be converted to the DICOM images. We once tried

to do that, and in order to do that you need the original PSL images to the DICOM images by using a specific program. Would it still be possible to run a study with conventional films rather than the phosphorus screens in order to increase the resolution of the images? If you use X-ray film, the film resolution is very, very high as compared with the imaging plate systems. At present, the imaging plate system allows the minimum resolution of 25 micrometer. That resolution is too much for the carbon-14 autoradiography because carbon-14 beta energy spreads around 100 micrometers or so in a tissue section. Basically, there is no reason for a resolution less than 100 micrometers, so in the case of the imaging plate system its enough. If you want to perform the autoradiography using the tritium, normally tritium autoradiography resolution is about 10 micrometers or so. The minimum for the imaging plate system is 25 micrometers, so if you want to perform the very, very fine imaging for the tritium autoradiography, still using x-ray film is advantageous versus the imaging plate system. It has been shown that directly after extending exposure a waiting time before scanning of 10-15 minutes is recommended due to a fast reduction of signal. This reduction is smaller after 15 minutes. Can you make any comments on that? Right after the exposure, you need to wait maybe five, ten minutes, but after the exposure sometimes they will sit in a cassette and be put in a lead safe (a shield box to reduce background radiation effect during contact exposure). When you take it out, if there is a temperature difference you need to wait five, ten minutes in order to reach the temperature equivalent. I have ever heard so from Fuji Film. In normal cases we take 5-10 minutes before reading imaging plates. Have you ever attempted to extract the slices with organic solvent to look for irreversible binding? Yes, there was once where I did, but that was the age of the x-ray films. Ive tried to remove the free material by using an organic solvent treatment, but in order to treat tissue sections, they should be transferred to the glass surface, as I mentioned before, because normally the tissue section is taken onto the scotch tape and the treatment of the organic solvent might cause a serious effect to the sections on the scotch tape. So, to perform the organic solvent treatment you need to transfer the sections onto the glass surface (which is difficult to perform). In MALDI imaging, is there a risk of delocalization of compounds when you spray the matrix on the desiccated section? Thats a problem, so I mentioned about IT TOF mass imaging. We perform the matrix application by airbrush spray. That will cause the diffusion of the material. But in the case of protein studies using the MALDI TOF imaging system, that is done by a chemical printer and a spot is like a circle spot and is separated only in each spot. So, the diffusion of material should not be considered. Resolution is not so good, but

each spot separated so the diffusion is practically no problem when we perform the MALDI TOF imaging. This question is for Nemoto-san. Can the client receive the tissue sections after the study is complete? Yes, if a client wants we can provide that. Is LSC sufficient and recommended for dog since it is a large animal, if rat will be analyzed by QWBA when both species are the non-clinical species of choice? I normally recommend to perform the WBA, not QWBA in order to decide the tissue for dissection and in order to confirm the presence of unexpected distribution. Even in the case of tissue dissection, which is sufficient for the data submission (to regulation authorities), I always recommend performing several autoradiography. How do you determine the limit of quantitation in QWBA? The limit of quantitation, the lower limit of quantitation can be judged by the size of the ROI and the PSL intensity. Weve analyzed relationships between the size of the ROI and the PSL intensity. Of course the CV value increases when the intensity of PSL is low. Also, the CV value increases when the size of the ROI is very small. Weve checked the CV values in various different ROI sizes and various different PSL intensities and weve found out the equation showing the CV of the region of interest is different in PSL intensity and the different sizes. By applying such an equation, we can determine the lower limit sensitivity. It is regulated by the PSL intensity and the size of the ROI. ROI means the region of interest.