Objectives: To educate about the methods used to perform Quantitative Whole-Body Autoradiography (QWBA) and Micro-Autoradiography (MARG) to facilitate an understanding of the benefits and limitations of the techniques. To present examples of how QWBA and MARG have been used to quantitatively and qualitatively evaluate drugs.

1. Eric Solon, Ph.D., QPS, LLC, Newark, Delaware, USA
THE USE OF MICRO- AND MACRO-
AUTORADIOGRAPHY TO STUDY THE TISSUE
DISTRIBUTION OF SMALL AND LARGE MOLECULES

2. Introduction
Objectives
To educate about the methods used to perform
Quantitative Whole-Body Autoradiography (QWBA)
and Micro-Autoradiography (MARG) to facilitate an
understanding of the benefits and limitations of the
techniques.
To present examples of how QWBA and MARG have
been used to quantitatively and qualitatively
evaluate drugs.
17 May 2013 Confidential 2

3. Introduction
Presentation Outline
Examples:
Definitive Tissue Distribution, PK, and Human
Radiation Dosimetry Estimations
Target Tissue and Tumor Penetration
Routes of Elimination
Adult and Fetal Brain Distribution and Metabolism of
14C-AZT
Brain Distribution and Efficacy of a 14C-siRNA
Distribution of 14C-Cyclodextrin in a Feline Niemann
Pick C Model
17 May 2013 Confidential 3

4. QWBA Methods
Study Design
17 May 2013 Confidential 4
Goal of QWBA is to provide tissue
concentration and spatial
distribution data to determine
Tissue Pharmacokinetics
All laboratory species may be used.
Long- & short-lived beta emitters
are used (e.g. 14C, 3H, 125I, 35S, 45Ca, 111In, 90Y)
Image Resolution is 25-100 µm
Tissue Concentration range
~ 0.0001 -10 µCi/g of tissue

5. QWBA Method
Technical Procedures
 Dose animals with radiolabeled
compound. IV, PO, SC, Intrathecal, direct
brain Infusion
 Blood Collection (for plasma
determinations)
 Euthanize animals at chosen time pts.
(~10 for reliable PK)
 Freeze and euthanize intact in hexane-
dry ice bath
 Embed carcass in
Carboxymethylcellulose
 Cryosection (~ 40μm) carcass at several
levels and dehydrate.
 Dehydrate Sections (2 days)
 Expose Sections and Calibration
Standards to Phosphor Imaging plates (in
lead box 4-days)
17 May 2013 Confidential 5

6. QWBA Method
Technical Procedures
 Scan Phosphor Imaging Plate & Digitally Image radioactivity in
tissues using phosphor image scanner (direct imaging or film also
possible).
 Image Analysis to Obtain Tissue Concentrations radioactivity in
tissues by image analysis. Densitometry is directly related to
concentration of radioactivity.
17 May 2013 Confidential 6

7. QWBA – Benefits and Limitations
Benefits
• In Situ Examination Preserves Spatial Distribution at Specified Time Points
• High Resolution Images (pixels = 25-100 µm)
• Quantitative (LLOQ ~ 2 DPM/mg, 2220 dpm/g, 44 dpm/0.5 cm2 )
• Obtain concentration data over days, weeks, months, and years
• Measure All Tissues (routine for >40 tissues) at any time.
17 May 2013 Confidential 7
Limitations
• Ex vivo
• Macro-Autoradiography Only Whole-Body Sections are not of histology quality
• Image Reflects Drug-Derived Radioactivity, i.e., parent drug and metabolites.
BUT the whole-body sections and/or residual tissues can be used with other
Bioanalytical techniques such as MALDI-MS and/or LC/MS/MS to identify
parent drug and/or metabolites!

8. Microautoradiography
(MARG)

9. MARG Technique
MARG
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MARG is a High Resolution Histological Tool to investigate spatial
localization of radiolabeled drugs at a tissue, and cellular level.
Ex vivo and exsanguination occurs
Numerous elaborations on the techniques
Old technique. The basic principals have remained unchanged for
> 40 years.
Cryo-preservation required for soluble compounds. Liquid tissue
fixation (formalin) often solubilizes and relocates diffusible test
articles. Exception for receptor-bound TA.
Not Quantitative – No standards used, prone to artifacts, lack of
control on detection media and section thickness.

10. MARG Technique
MARG Procedures
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Dose animals with radiolabeled compounds
Euthanize animals at chosen time points
Necropsy to remove sample tissues
Trim to 5 x 5 mm2
Snap-freeze onto stub in Nliq-cooled isopentane
Cryosection tissue (~ 4-10 µm) and thaw mount onto slides pre-coated
with photo-emulsion. UNDER DARKROOM CONDITIONS!
Expose in a light-tight slide box with dessicant at 4ºC for 1-100 days
depending on radioconcentration.
Develop, Stain, Examine under microscope. Immunohistochemical
stains may be used ( co-localize receptors/targets) but method
development needed due to possible effects of photographic emulsion
on antibody binding

11. MARG Technique
Topic Title
17 May 2013 Confidential 11

12. QWBA & MARG
Examples

13. Tissue concentration data routinely obtained for >40
tissues.
Plasma & Tissue PK parameters are determined.
Example: Definitive Tissue Distribution & PK
Mean - µg equiv/g tissue
Tissue Type Tissue 1 h 3 h 6 h 12 h 24 h 48 h 72 h 168 h 696 h
Vascular/ Lymphatic Blood (cardiac) 2.249 2.979 1.824 1.268 0.794 0.090 0.019 BQL BQL
Vascular/ Lymphatic Bone Marrow 6.128 7.666 6.621 4.914 2.379 0.362 0.038 BQL BQL
Vascular/ Lymphatic Lymph Node 3.704 6.340 6.961 4.698 2.666 0.286 0.048 BQL BQL
Vascular/ Lymphatic Spleen 9.638 8.130 8.929 6.263 3.273 0.368 0.074 BQL BQL
Vascular/ Lymphatic Thymus 2.471 6.862 7.160 6.076 2.992 0.272 BQL BQL BQL
Excretory/ Metabolic Bile (in duct) 63.479 189.946 116.627 70.668 19.480 11.669 BQL BQL BQL
Excretory/ Metabolic Renal Cortex 14.790 12.692 18.409 10.299 6.864 1.069 0.212 0.027 BQL
Excretory/ Metabolic Renal Medulla 12.220 10.331 14.111 6.799 6.261 0.713 0.106 BQL BQL
Excretory/ Metabolic Liver 40.766 32.326 39.866 31.911 19.397 4.076 0.362 0.067 BQL
Excretory/ Metabolic Urinary Bladder 2.132 13.686 6.673 4.497 2.106 0.202 0.034 BQL BQL
Excretory/ Metabolic Urinary Bladder (contents) 0.237 6.773 8.426 1.670 2.648 0.097 BQL BQL BQL
Central Nervous System Brain (cerebellum) 2.418 9.831 13.049 11.227 7.788 1.336 0.084 BQL BQL
Central Nervous System Cerebellum (gray matter) 2.667 10.811 13.166 10.313 6.217 0.818 BQL BQL BQL
Central Nervous System Cerebellum (white matter) 1.266 6.890 9.407 17.281 13.017 6.877 0.317 BQL BQL
Central Nervous System Brain (cerebrum) 2.079 8.310 12.027 11.108 8.209 1.179 0.064 BQL BQL
Central Nervous System Cerebrum (gray matter) 2.162 8.893 12.673 9.996 6.712 0.686 0.076 BQL BQL
Central Nervous System Cerebrum (white matter) 1.273 6.911 9.862 13.472 12.931 3.662 BQL BQL BQL
Central Nervous System Brain (medulla) 2.332 11.082 17.022 19.139 12.163 1.469 0.137 BQL BQL
Central Nervous System Medulla (gray matter) 2.714 13.880 18.249 16.110 8.979 2.620 0.082 BQL BQL
Central Nervous System Medulla (white matter) 2.462 11.283 14.296 20.418 12.764 1.467 0.243 BQL BQL
Central Nervous System Spinal Cord 2.116 9.371 12.184 19.287 13.767 4.367 0.448 0.067 BQL
Central Nervous System Spinal Cord (gray matter) 2.689 12.328 18.009 19.394 12.107 1.893 0.160 0.019 BQL
Central Nervous System Spinal Cord (white matter) 0.718 4.316 7.648 14.342 8.387 7.976 0.936 0.186 BQL
Endocrine Adrenal Gland 23.128 26.293 26.886 16.296 9.636 1.398 0.113 BQL BQL
Endocrine Pituitary Gland 8.864 14.339 14.347 9.661 6.201 0.478 0.034 BQL BQL
Endocrine Thyroid 10.412 12.119 12.431 8.833 3.783 0.444 0.029 BQL BQL
Secretory Harderian Gland 4.140 16.081 27.762 13.783 12.120 1.337 0.067 BQL BQL
Secretory Mammary Gland Region 1.831 2.399 1.899 1.219 0.884 0.132 0.063 BQL BQL
Secretory Pancreas 12.113 16.042 13.081 9.462 6.230 0.678 0.046 BQL BQL
Secretory Salivary Gland 9.264 16.128 10.912 8.946 6.169 0.621 0.028 BQL BQL
Fatty Adipose (brown) 6.027 10.026 9.698 7.392 4.891 1.198 0.247 BQL BQL
Fatty Adipose (white) 0.801 1.206 1.314 0.601 0.290 0.069 0.067 BQL BQL
Dermal Skin (non-pigmented) 1.830 4.011 2.739 2.826 1.387 0.270 BQL BQL BQL
Dermal Skin (pigmented) 1.379 3.183 3.399 3.137 1.260 0.222 0.099 0.073 BQL
Reproductive Epididymis 0.908 3.742 6.436 6.261 6.113 0.763 0.079 BQL BQL
Reproductive Prostate Gland 1.498 4.763 6.060 4.306 4.863 0.418 BQL BQL BQL
Reproductive Seminal Vesicles 1.260 3.284 3.198 2.762 1.349 0.166 BQL BQL BQL
Reproductive Testis 0.490 2.383 4.746 6.136 4.288 0.936 0.094 BQL BQL
Skeletal/Muscular Bone 0.191 0.136 0.749 0.494 0.093 0.036 BQL BQL BQL
Skeletal/Muscular Heart (myocardium) 14.338 16.461 11.706 8.661 6.419 0.624 0.037 BQL BQL
Skeletal/Muscular Skeletal Muscle 3.264 6.136 6.921 4.446 2.622 0.266 0.047 BQL BQL
Respiratory Tract Lung 8.946 9.296 6.243 6.966 3.617 0.364 BQL BQL BQL
Alimentary Canal Cecum 7.810 6.136 17.614 21.636 10.783 2.661 0.060 BQL BQL
Alimentary Canal Cecum (contents) 0.102 109.220 *362.778 213.726 194.927 16.964 0.480 BQL BQL
Alimentary Canal Esophagus 12.676 6.606 2.788 3.636 1.926 0.207 0.062 BQL BQL
Alimentary Canal Large Intestine 6.721 11.346 9.367 31.210 37.176 0.696 0.071 BQL BQL
Alimentary Canal Large Intestine (contents) 0.026 0.624 1.128 289.034 *688.776 21.300 1.028 BQL BQL
Alimentary Canal Oral Mucosa 2.141 3.483 3.326 2.294 1.262 0.264 BQL BQL BQL
Alimentary Canal Small Intestine *313.169 19.696 33.212 13.466 12.467 0.816 0.102 BQL BQL
Alimentary Canal Small Intestine (contents) 179.406 166.660 229.973 43.061 72.623 4.004 0.488 0.026 BQL
Alimentary Canal Stomach (gastric mucosa) 31.624 38.316 32.960 29.717 16.862 1.646 0.109 BQL BQL
Alimentary Canal Stomach (contents) *663.461 143.669 139.700 6.011 6.777 0.413 0.044 BQL BQL
Ocular Eye (lens) 0.032 0.131 0.282 0.291 0.211 0.136 0.041 BQL BQL
Ocular Eye (uveal tract) 3.118 9.081 14.607 11.442 6.469 4.392 1.289 0.638 0.468

14. Autoradiographs showing
the tissue distribution in
albino (Sprague-Dawley)
and pigmented rats (Long
Evans).
Note the amount of
radioactivity in the eye of
the Long Evans rat vs. the
Sprague-Dawley rat.
Data is routinely used to
determine Human
Radiation Dosimetry in
>40 tissues.
Example: Definitive TD and Tissue PK

15. 5/17/2013 15
Example: Radiation Dosimetry
Human radiolabeled drug studies are performed as part of Phase II clinical
trials to determine human metabolism and pharmacokinetics of new drug
entities. 14C-and 3H-labeled compounds are routinely used
Dosimetry predictions rely on mathematical models and radioactive
tissue/organ concentration and/or excretion data, which are obtained from
radioactive dosing animal studies (typically, rodents).
Various methods to determine human and dosimetry predictions have
been published by FDA and the International Commission on Radiation
Protection (ICRP), but the calculations and data obtained from the various
methods can produce different predictions.
Different Pharma companies and CROs have developed different methods
over the years and some being used today are outdated and/or
inappropriate when using QWBA data and 14C or 3H.

16. 5/17/2013 16
Hendee-Marinelli MIRD ICRP w/out W.F.
D (rem) Dman (rem) mSV rem
Adipose (brown) 0.02554 0.21449 0.09320 0.00932
Adipose (white) 0.00845 0.16601 0.07213 0.00721
Adrenal Gland 0.08143 0.28389 0.12335 0.01234
Blood (cardiac) 0.01904 0.06137 0.02667 0.00267
Bone (femur) 0.00576 0.17537 0.07620 0.00762
Bone Marrow (femur) 0.02236 0.09351 0.04063 0.00406
Brain 0.00259 0.06318 0.02745 0.00275
Cecum 0.02854 0.38506 0.16731 0.01673
Epididymis 0.05857 0.22230 0.09659 0.00966
Eye Lens 0.00585 0.04834 0.02100 0.00210
Eye Uveal Tract 0.02214 0.35203 0.15296 0.01530
Heart 0.07229 0.19932 0.08660 0.00866
Large Intestine 0.11983 0.59201 0.25724 0.02572
Liver 0.18987 0.17898 0.07777 0.00778
Lung 7.17978 20.63142 8.96459 0.89646
Lymph Node 0.03693 0.19621 0.08525 0.00853
Pancreas 0.05170 0.19386 0.08423 0.00842
Pituitary Gland 0.06679 0.20283 0.08813 0.00881
Prostate Gland 0.03461 0.24700 0.10733 0.01073
Renal Cortex 0.42589 0.54124 0.23518 0.02352
Renal Medulla 0.17270 0.43635 0.18960 0.01896
Salivary Gland 0.02316 0.10613 0.04612 0.00461
Seminal Vesicles 0.02526 0.19243 0.08361 0.00836
Skeletal Muscle 0.02067 0.20226 0.08788 0.00879
Skin 0.02868 0.25401 0.11037 0.01104
Small Intestine 0.02063 0.30142 0.13097 0.01310
Spinal Cord 0.01892 0.31804 0.13819 0.01382
Spleen 0.11811 0.26489 0.11510 0.01151
Stomach (gastric
mucosa) 3.17550 6.73324 2.92567 0.29257
Testis 0.00789 0.06870 0.02985 0.00298
Thymus 0.00970 0.07178 0.03119 0.00312
Thyroid 0.02839 0.21398 0.09298 0.00930
Urinary Bladder 0.38319 0.73106 0.31765 0.03177
Whole Body Total 12.49077 0.72000 1.52825 0.15282
Example: Radiation Dosimetry
Conclusions of a Comparison:
Different calculations can
produce different predictions of
radiation exposure.
Some calculations (i.e. GI
transit model), which are
developed for penetrating
radiation (e.g., PET, Spect,
Gamma Scintigraphy) are not
appropriate for predicting 14C
and 3H exposures and can over
estimate actual tissue
exposure.

17. Examples – Tumor Penetration
Enables the distinction between the necrotic and solid portions of
a tumor that can have very different concentrations.
Provides a way to see if there are other potential therapeutic
targets for the compound by determining concentrations in other
tissues.

18. Examples – Ocular Drug Distribution
Phosphor imaging and MARG can be used to
examine quantitative distribution of radiolabeled
compounds in fine ocular structures of rats, rabbits,
and dogs.

19. Example: Routes of Elimination
Autoradiograph of bile-duct
cannulated rats given an IV
dose of a 14C-labeled drug
QWBA demonstrated
intestinal secretion as an
unanticipated route of
elimination

20. Adult and Fetal Brain Distribution and
Metabolism of 14C-AZT
Background and Study Design
17 May 2013 Confidential 20
Proof of principal study on Placental Transfer to demonstrate the utility of QWBA for
this examination.
Combination study to examine fetal and maternal tissue distribution of
14C-azidothymidine (14C-AZT) after a single intravenous administration to a pregnant
female rat.
QWBA revealed differential distribution of 14C-AZT-derived radioactivity in fetal and
maternal, brain and liver. Concentrations of radioactivity in fetal brain and liver were
higher than in the adult.
Fetal and maternal brain and liver were obtained by necropsy of an additional
pregnant rat for MARG and metabolite profiling by radio-HPLC.
To further characterize the different patterns of distribution, samples of fetal and
maternal brain and liver were homogenized, extracted and analyzed by radio-HPLC to
obtain a metabolite profile of each tissue and differences were identified.
Further analysis using mass spectroscopy techniques provided identification of these
metabolites.

21. Adult and Fetal Brain Distribution and
Metabolism of 14C-AZT
QWBA Results
Whole-body Autoradioluminographs of an pregnant rat (day
17) (left) and a 17-day old fetus (right) showing differential
distribution of 14C-AZT-derived radioactivity in liver and brain.
Where is it at the cellular level?
Is this compound AZT or Metabolite?
17 May 2013 Confidential 21

22. Adult and Fetal Brain Distribution and
Metabolism of 14C-AZT
Micro-Autoradiography in Brain and Liver
Photomicroautoradiographs of the cellular localization of 14C-AZT-derived
radioactivity in the brain and liver of a pregnant rat (top left and right respectively)
and in the brain and liver of a 17-day old fetus (bottom left and right respectively).
(Hematoxylin & Eosin Stain, 400X; ML = Molecular Layer, GL = Granular Layer, WM
= White Matter, PC = Purkinje Cells) .
17 May 2013 Confidential 22
Maternal Tissue
Fetal Tissue
Brain Liver

23. Adult and Fetal Brain Distribution and
Metabolism of 14C-AZT
AZT Metabolism
Radiochromatographs showing the metabolite profiles obtained from
maternal and fetal liver and brain samples after a single intravenous
administration of 14C-AZT.
17 May 2013 Confidential 23
Liver:
Brain:

24. Brain Distribution and Efficacy of a 14C-siRNA for
Huntington’s Disease
Background and Study Design
Huntington’s disease is caused by a n overexpression of the CAG repeat
in the Huntington gene (Htt). Normally, this section of DNA is repeated 10
to 28 times. But in persons with Huntington's disease, it is repeated 36 to
120 times.
The Sponsor of this study worked with QPS to study the distribution and
efficacy of an administered 14C-siRNA in rats.
14C-siRNA was directly infused into the striatum of rat brains over time
periods up to 7 days.
Each brain was removed at different time points after dosing and were
frozen and sectioned for quantitative autoradiography analysis and
analysis of tissue punches by real-time PCR.
17 May 2013 Confidential 24

25. Brain Distribution and Efficacy of a 14C-siRNA
Dosing and Sample Collection
Sprague Dawley Rats were fitted with indwelling canulas that were
stereotaxically positioned into the striatum of the brain.
14C-siRNA was infused into the striatum at slow rates over times up to 7
days.
The brain was removed and flask frozen on dry ice, and plasma was
collected.
17 May 2013 Confidential 25

26. Brain Distribution and Efficacy of a 14C-siRNA
Quantitative Autoradioluminography in Brain
Frozen Brains were cryosectioned
through the striatal region at 40 µm
thickness, sections were collected onto
glass slides, and immediately dried on a
slide warmer.
Brain sections were exposed to
phosphor imaging plates along with 14C
calibration standards for 4 days and the
imaging plates were scanned at a
resolution of 50 µm.
Brain concentrations were determined
at discreet locations throughout the
brain to create a detailed histogram of
concentrations through the injection
site.
17 May 2013 Confidential 26

27. Brain Distribution and Efficacy of a 14C-siRNA
Quantitative Autoradioluminography in Brain
17 May 2013 Confidential 27

28. Brain Distribution and Efficacy of a 14C-siRNA
Quantitative Real-Time PCR in Brain
Brain Punch samples were collected from various regions in sections that
were adjacent to those collected for autoradiography during
cryosectioning
Samples were analyzed by rtPCR
Results showed that the Htt gene was silenced.
17 May 2013 Confidential 28
Sample Detector Ct ∆ Ct
Avg
∆Ct
∆Ct
SD
∆Ct
%CV
Rat #1_300u_2 GAPDH 26.4692
7.0702
7.6658 0.7816 10.1961
Rat #1_300u_2 Htt 33.5393
Rat #1_300u_2 GAPDH 26.4108
8.5509Rat #1_300u_2 Htt 34.9616
Rat #1_300u_2 GAPDH 26.5414
7.3764Rat #1_300u_2 Htt 33.9178

29. Distribution of 14C-Cyclodextrin in Feline
Neiman-Pick C Model
Background
Niemann-Pick Disease Type C is caused by an accumulation of materials
(cholesterol and other fatty acids) in the body's cells that leads to
progressive intellectual decline, loss of motor skills, seizures and
dementia. The disease progresses at varying rates. Young children who
display neurological symptoms generally have an aggressive form of the
disease, while others may not display symptoms for years.
The Sponsors of this study (Jansen R&D, LLC, and University of
Pennsylvania) worked with QPS to study the distribution of an
administered 14C-Cyclodextrin in a Feline Niemann Pick Model to
characterize the spatial distribution and pharmacokinetics in the central
nervous system and other organs to gain a better understanding for the
treatment of the disease in Humans.
17 May 2013 Confidential 29

30. Distribution of 14C-Cyclodextrin in Feline
Neiman-Pick C Model
Study Design
Female Niemann-Pick Cats (Univ. of Penn) were
administered a single intrathecal dose of 14C-Cyclodextrin at
120 mg/cat (200 µCi/cat)
One cat per time point was euthanized at 0.25 h, 1 h, 4 h, 8
h, 12 h, and 24 h post-dose, and each carcass was frozen for
QWBA analysis.
Concentrations of Cyclodextrin were determined in > 30
tissues including discreet regions of the brain.
17 May 2013 Confidential 30

31. Distribution of 14C-Cyclodextrin in Feline
Neiman-Pick C Model
Results
17 May 2013 Confidential 31
8 h 12 h 24 h

32. Distribution of 14C-Cyclodextrin in Feline
Neiman-Pick C Model
Results
Tissue PK Parameters of Cyclodextrin (µg/g tissue) in Niemann-Pick Cats
17 May 2013 Confidential 32
Tissue AUCall AUCinf_obs Cmax Tmax T1/2
# of pt.s in
T1/2 r2
ug equiv·h/g ug equiv·h/g ug equiv/g h h
Adrenal Gland 205.470 250.919 32.709 0.25 10.097 3 0.93
Blood (cardiac) 140.164 159.866 40.011 1 10.8 4 0.17
Brain (cerebellum) (hi) 3620.677 4072.258 689.516 1 6.3 5 0.53
Brain (cerebellum) (low) 405.069 Missing 22.485 24 Missing 0 Missing
Brain (cerebrum) (hi) 3184.710 5686.564 417.944 4 32.6 3 0.78
Brain (cerebrum) (low) 365.502 621.691 25.759 12 13.0 2 1.00
Brain (medulla) 969.982 1089.337 116.422 4 6.8 2 1.00
Kidney Cortex 484.666 Missing 33.101 24 Missing 0 Missing
Liver 65.103 106.111 12.267 1 ND 5 0.22
Nasal Turbinates 3163.073 3952.117 1031.483 0.25 17.555 3 0.90
Pituitary Gland 2278.187 2406.100 426.351 0.25 ND 4 0.61
Skeletal Muscle 16.341 21.900 2.791 1 ND 3 0.60
Spinal Cord 1782.007 2105.836 214.885 4 8.6 4 0.57
Urinary Bladder 107.586 235.612 15.243 1 ND 4 0.62
ND = Not Determined due to insufficient data

33. Further Image analysis
provided a histogram of
Brain concentrations
from which data was
extracted to obtain PK
parameters at discreet
locations throughout
the Brain
17 May 2013 Confidential 33
Distribution of 14C-Cyclodextrin in Feline
Neiman-Pick C Model
Results

34. Distribution of 14C-Cyclodextrin in Feline
Neiman-Pick C Model
Conclusions
Drug-derived radioactivity was absorbed from the cerebellomedulary cistern and was
widely distributed to tissues of the cats after a single intrathecal dose of [14C]Cyclodextrin.
Visual examination of the autoradiographs showed that while concentrations in blood and
most other tissues were decreasing, penetration of drug-derived radioactivity into the
deeper parts of the CNS tissues was ongoing and concentrations in different regions of the
brain varied over 24 h.
Tissues, besides the CNS, with the highest concentrations (≥ 100 µg equiv/g) of
radioactivity were nasal turbinates (1031.5 µg equiv/g at 0.25 h), and pituitary gland
(426.4 µg equiv/g at 0.25 h).
High concentrations were also present in the contents of the urinary bladder
(2842.8 µg equiv/g at 4 h), which demonstrated that renal excretion was the major route
of elimination.
Prolonged exposure of tissues that are outside of the CNS is expected, albeit at low
concentrations, as drug-derived radioactivity is eliminated from the CNS compartment and
then eliminated from the body.
17 May 2013 Confidential 34

35. Overall Conclusions
QWBA provides detailed, discreet, quantitative, tissue
distribution and detailed PK information for small and large
molecule drugs.
MARG provides detailed, discreet, qualitative, cellular
distribution information for small and large molecule drugs.
Several other analytical techniques such as LC/MS/MS, and
rtPCR can be easily combined to provide a wealth of
knowledge regarding the detailed distribution, concentration
and kinetics of various test drugs in the central and peripheral
nervous system of laboratory animals.
17 May 2013 Confidential 35

36. Acknowledgements
QPS
Alfred Lordi
Paul Strzemienski
Tony Srnka
Jackie Morgan
Jackie Eckbold
Sarah Patterson
Yvette Warner
Martin Hulse
Marna DiOssi
Helen Shen
Zamas Lam
Ben Chien
17 May 2013 Confidential 36
Charles Vite, DVM, Ph.D.
Janssen Research
& Development, L.L.C.
University
of Pennsylvania
Mark Kao, Ph.D.