THE USE OF MICRO- AND MACRO-AUTORADIOGRAPHY TO STUDY THE TISSUE DISTRIBUTION OF SMALL AND LARGE...

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

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

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


QWBA Methods

Study Design


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

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)


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.


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.


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!

Microautoradiography (MARG)

MARG Technique

MARG


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.

MARG Technique

MARG Procedures


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

MARG Technique

Topic Title


QWBA & MARG Examples

Tissue concentration data routinely obtained for >40 tissues. Plasma & Tissue PK parameters are determined.

Example: Definitive Tissue Distribution & PK

Mean - µg equiv/g tissueTissue 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 BQLVascular/ Lymphatic Bone Marrow 6.128 7.666 6.621 4.914 2.379 0.362 0.038 BQL BQLVascular/ Lymphatic Lymph Node 3.704 6.340 6.961 4.698 2.666 0.286 0.048 BQL BQLVascular/ Lymphatic Spleen 9.638 8.130 8.929 6.263 3.273 0.368 0.074 BQL BQLVascular/ Lymphatic Thymus 2.471 6.862 7.160 6.076 2.992 0.272 BQL BQL BQLExcretory/ Metabolic Bile (in duct) 63.479 189.946 116.627 70.668 19.480 11.669 BQL BQL BQLExcretory/ Metabolic Renal Cortex 14.790 12.692 18.409 10.299 6.864 1.069 0.212 0.027 BQLExcretory/ Metabolic Renal Medulla 12.220 10.331 14.111 6.799 6.261 0.713 0.106 BQL BQLExcretory/ Metabolic Liver 40.766 32.326 39.866 31.911 19.397 4.076 0.362 0.067 BQLExcretory/ Metabolic Urinary Bladder 2.132 13.686 6.673 4.497 2.106 0.202 0.034 BQL BQLExcretory/ 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 BQLCentral Nervous System Cerebellum (gray matter) 2.667 10.811 13.166 10.313 6.217 0.818 BQL BQL BQLCentral Nervous System Cerebellum (white matter) 1.266 6.890 9.407 17.281 13.017 6.877 0.317 BQL BQLCentral Nervous System Brain (cerebrum) 2.079 8.310 12.027 11.108 8.209 1.179 0.064 BQL BQLCentral Nervous System Cerebrum (gray matter) 2.162 8.893 12.673 9.996 6.712 0.686 0.076 BQL BQLCentral Nervous System Cerebrum (white matter) 1.273 6.911 9.862 13.472 12.931 3.662 BQL BQL BQLCentral Nervous System Brain (medulla) 2.332 11.082 17.022 19.139 12.163 1.469 0.137 BQL BQLCentral Nervous System Medulla (gray matter) 2.714 13.880 18.249 16.110 8.979 2.620 0.082 BQL BQLCentral Nervous System Medulla (white matter) 2.462 11.283 14.296 20.418 12.764 1.467 0.243 BQL BQLCentral Nervous System Spinal Cord 2.116 9.371 12.184 19.287 13.767 4.367 0.448 0.067 BQLCentral Nervous System Spinal Cord (gray matter) 2.689 12.328 18.009 19.394 12.107 1.893 0.160 0.019 BQLCentral 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 BQLEndocrine Pituitary Gland 8.864 14.339 14.347 9.661 6.201 0.478 0.034 BQL BQLEndocrine Thyroid 10.412 12.119 12.431 8.833 3.783 0.444 0.029 BQL BQLSecretory Harderian Gland 4.140 16.081 27.762 13.783 12.120 1.337 0.067 BQL BQLSecretory Mammary Gland Region 1.831 2.399 1.899 1.219 0.884 0.132 0.063 BQL BQLSecretory Pancreas 12.113 16.042 13.081 9.462 6.230 0.678 0.046 BQL BQLSecretory 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 BQLFatty 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 BQLDermal 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 BQLReproductive Prostate Gland 1.498 4.763 6.060 4.306 4.863 0.418 BQL BQL BQLReproductive Seminal Vesicles 1.260 3.284 3.198 2.762 1.349 0.166 BQL BQL BQLReproductive 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 BQLRespiratory Tract Lung 8.946 9.296 6.243 6.966 3.617 0.364 BQL BQL BQLAlimentary Canal Cecum 7.810 6.136 17.614 21.636 10.783 2.661 0.060 BQL BQLAlimentary Canal Cecum (contents) 0.102 109.220 *362.778 213.726 194.927 16.964 0.480 BQL BQLAlimentary Canal Esophagus 12.676 6.606 2.788 3.636 1.926 0.207 0.062 BQL BQLAlimentary Canal Large Intestine 6.721 11.346 9.367 31.210 37.176 0.696 0.071 BQL BQLAlimentary Canal Large Intestine (contents) 0.026 0.624 1.128 289.034 *688.776 21.300 1.028 BQL BQLAlimentary Canal Oral Mucosa 2.141 3.483 3.326 2.294 1.262 0.264 BQL BQL BQLAlimentary Canal Small Intestine *313.169 19.696 33.212 13.466 12.467 0.816 0.102 BQL BQLAlimentary Canal Small Intestine (contents) 179.406 166.660 229.973 43.061 72.623 4.004 0.488 0.026 BQLAlimentary Canal Stomach (gastric mucosa) 31.624 38.316 32.960 29.717 16.862 1.646 0.109 BQL BQLAlimentary 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 BQLOcular Eye (uveal tract) 3.118 9.081 14.607 11.442 6.469 4.392 1.289 0.638 0.468

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

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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.

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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.

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.

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.

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

Adult and Fetal Brain Distribution and Metabolism of 14C-AZT

Background and Study Design


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.


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?



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) .


Maternal Tissue

Fetal Tissue

Brain Liver


AZT Metabolism Radiochromatographs showing the metabolite profiles obtained from

maternal and fetal liver and brain samples after a single intravenous administration of 14C-AZT.


Liver:

Brain:

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.


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.



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.



Quantitative Autoradioluminography in Brain



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.


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

8.5509 Rat #1_300u_2 Htt 34.9616

Rat #1_300u_2 GAPDH 26.5414

7.3764 Rat #1_300u_2 Htt 33.9178

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.



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.



Results


8 h

12 h

24 h


Results Tissue PK Parameters of Cyclodextrin (µg/g tissue) in Niemann-Pick Cats


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

Further Image analysis provided a histogram of Brain concentrations from which data was extracted to obtain PK parameters at discreet locations throughout the Brain



Results


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.


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.


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


Charles Vite, DVM, Ph.D.

Janssen Research & Development, L.L.C.

University of Pennsylvania

Mark Kao, Ph.D.

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THE USE OF MICRO- AND MACRO-AUTORADIOGRAPHY TO STUDY THE TISSUE DISTRIBUTION OF SMALL AND LARGE...

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