Eric Solon, Ph.D., QPS, LLC, Newark, Delaware, USATHE USE OF MICRO- AND MACRO-AUTORADIOGRAPHY TO STUDY THE TISSUEDISTRIBUT...
IntroductionObjectivesTo educate about the methods used to performQuantitative Whole-Body Autoradiography (QWBA)and Micro-...
IntroductionPresentation OutlineExamples:Definitive Tissue Distribution, PK, and HumanRadiation Dosimetry EstimationsTarge...
QWBA MethodsStudy Design17 May 2013 Confidential 4Goal of QWBA is to provide tissueconcentration and spatialdistribution d...
QWBA MethodTechnical Procedures Dose animals with radiolabeledcompound. IV, PO, SC, Intrathecal, directbrain Infusion Bl...
QWBA MethodTechnical Procedures Scan Phosphor Imaging Plate & Digitally Image radioactivity intissues using phosphor imag...
QWBA – Benefits and LimitationsBenefits• In Situ Examination Preserves Spatial Distribution at Specified Time Points• High...
Microautoradiography(MARG)
MARG TechniqueMARG17 May 2013 Confidential 9MARG is a High Resolution Histological Tool to investigate spatiallocalization...
MARG TechniqueMARG Procedures17 May 2013 Confidential 10Dose animals with radiolabeled compoundsEuthanize animals at chose...
MARG TechniqueTopic Title17 May 2013 Confidential 11
QWBA & MARGExamples
Tissue concentration data routinely obtained for >40tissues.Plasma & Tissue PK parameters are determined.Example: Definiti...
Autoradiographs showingthe tissue distribution inalbino (Sprague-Dawley)and pigmented rats (LongEvans).Note the amount ofr...
5/17/2013 15Example: Radiation DosimetryHuman radiolabeled drug studies are performed as part of Phase II clinicaltrials t...
5/17/2013 16Hendee-Marinelli MIRD ICRP w/out W.F.D (rem) Dman (rem) mSV remAdipose (brown) 0.02554 0.21449 0.09320 0.00932...
Examples – Tumor PenetrationEnables the distinction between the necrotic and solid portions ofa tumor that can have very d...
Examples – Ocular Drug DistributionPhosphor imaging and MARG can be used toexamine quantitative distribution of radiolabel...
Example: Routes of EliminationAutoradiograph of bile-ductcannulated rats given an IVdose of a 14C-labeled drugQWBA demonst...
Adult and Fetal Brain Distribution andMetabolism of 14C-AZTBackground and Study Design17 May 2013 Confidential 20Proof of ...
Adult and Fetal Brain Distribution andMetabolism of 14C-AZTQWBA ResultsWhole-body Autoradioluminographs of an pregnant rat...
Adult and Fetal Brain Distribution andMetabolism of 14C-AZTMicro-Autoradiography in Brain and LiverPhotomicroautoradiograp...
Adult and Fetal Brain Distribution andMetabolism of 14C-AZTAZT MetabolismRadiochromatographs showing the metabolite profil...
Brain Distribution and Efficacy of a 14C-siRNA forHuntington’s DiseaseBackground and Study DesignHuntington’s disease is c...
Brain Distribution and Efficacy of a 14C-siRNADosing and Sample CollectionSprague Dawley Rats were fitted with indwelling ...
Brain Distribution and Efficacy of a 14C-siRNAQuantitative Autoradioluminography in BrainFrozen Brains were cryosectionedt...
Brain Distribution and Efficacy of a 14C-siRNAQuantitative Autoradioluminography in Brain17 May 2013 Confidential 27
Brain Distribution and Efficacy of a 14C-siRNAQuantitative Real-Time PCR in BrainBrain Punch samples were collected from v...
Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelBackgroundNiemann-Pick Disease Type C is caused by an accumul...
Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelStudy DesignFemale Niemann-Pick Cats (Univ. of Penn) wereadmi...
Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelResults17 May 2013 Confidential 318 h 12 h 24 h
Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelResultsTissue PK Parameters of Cyclodextrin (µg/g tissue) in ...
Further Image analysisprovided a histogram ofBrain concentrationsfrom which data wasextracted to obtain PKparameters at di...
Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelConclusionsDrug-derived radioactivity was absorbed from the c...
Overall ConclusionsQWBA provides detailed, discreet, quantitative, tissuedistribution and detailed PK information for smal...
AcknowledgementsQPSAlfred LordiPaul StrzemienskiTony SrnkaJackie MorganJackie EckboldSarah PattersonYvette WarnerMartin Hu...
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THE USE OF MICRO- AND MACRO-AUTORADIOGRAPHY TO STUDY THE TISSUE DISTRIBUTION OF SMALL AND LARGE MOLECULES

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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.

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

  1. 1. Eric Solon, Ph.D., QPS, LLC, Newark, Delaware, USATHE USE OF MICRO- AND MACRO-AUTORADIOGRAPHY TO STUDY THE TISSUEDISTRIBUTION OF SMALL AND LARGE MOLECULES
  2. 2. IntroductionObjectivesTo educate about the methods used to performQuantitative Whole-Body Autoradiography (QWBA)and Micro-Autoradiography (MARG) to facilitate anunderstanding of the benefits and limitations of thetechniques.To present examples of how QWBA and MARG havebeen used to quantitatively and qualitativelyevaluate drugs.17 May 2013 Confidential 2
  3. 3. IntroductionPresentation OutlineExamples:Definitive Tissue Distribution, PK, and HumanRadiation Dosimetry EstimationsTarget Tissue and Tumor PenetrationRoutes of EliminationAdult and Fetal Brain Distribution and Metabolism of14C-AZTBrain Distribution and Efficacy of a 14C-siRNADistribution of 14C-Cyclodextrin in a Feline NiemannPick C Model17 May 2013 Confidential 3
  4. 4. QWBA MethodsStudy Design17 May 2013 Confidential 4Goal of QWBA is to provide tissueconcentration and spatialdistribution data to determineTissue PharmacokineticsAll laboratory species may be used.Long- & short-lived beta emittersare used (e.g. 14C, 3H, 125I, 35S, 45Ca, 111In, 90Y)Image Resolution is 25-100 µmTissue Concentration range~ 0.0001 -10 µCi/g of tissue
  5. 5. QWBA MethodTechnical Procedures Dose animals with radiolabeledcompound. IV, PO, SC, Intrathecal, directbrain Infusion Blood Collection (for plasmadeterminations) Euthanize animals at chosen time pts.(~10 for reliable PK) Freeze and euthanize intact in hexane-dry ice bath Embed carcass inCarboxymethylcellulose Cryosection (~ 40μm) carcass at severallevels and dehydrate. Dehydrate Sections (2 days) Expose Sections and CalibrationStandards to Phosphor Imaging plates (inlead box 4-days)17 May 2013 Confidential 5
  6. 6. QWBA MethodTechnical Procedures Scan Phosphor Imaging Plate & Digitally Image radioactivity intissues using phosphor image scanner (direct imaging or film alsopossible). Image Analysis to Obtain Tissue Concentrations radioactivity intissues by image analysis. Densitometry is directly related toconcentration of radioactivity.17 May 2013 Confidential 6
  7. 7. QWBA – Benefits and LimitationsBenefits• 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 7Limitations• 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 otherBioanalytical techniques such as MALDI-MS and/or LC/MS/MS to identifyparent drug and/or metabolites!
  8. 8. Microautoradiography(MARG)
  9. 9. MARG TechniqueMARG17 May 2013 Confidential 9MARG is a High Resolution Histological Tool to investigate spatiallocalization of radiolabeled drugs at a tissue, and cellular level.Ex vivo and exsanguination occursNumerous elaborations on the techniquesOld technique. The basic principals have remained unchanged for> 40 years.Cryo-preservation required for soluble compounds. Liquid tissuefixation (formalin) often solubilizes and relocates diffusible testarticles. Exception for receptor-bound TA.Not Quantitative – No standards used, prone to artifacts, lack ofcontrol on detection media and section thickness.
  10. 10. MARG TechniqueMARG Procedures17 May 2013 Confidential 10Dose animals with radiolabeled compoundsEuthanize animals at chosen time pointsNecropsy to remove sample tissuesTrim to 5 x 5 mm2Snap-freeze onto stub in Nliq-cooled isopentaneCryosection tissue (~ 4-10 µm) and thaw mount onto slides pre-coatedwith photo-emulsion. UNDER DARKROOM CONDITIONS!Expose in a light-tight slide box with dessicant at 4ºC for 1-100 daysdepending on radioconcentration.Develop, Stain, Examine under microscope. Immunohistochemicalstains may be used ( co-localize receptors/targets) but methoddevelopment needed due to possible effects of photographic emulsionon antibody binding
  11. 11. MARG TechniqueTopic Title17 May 2013 Confidential 11
  12. 12. QWBA & MARGExamples
  13. 13. Tissue concentration data routinely obtained for >40tissues.Plasma & Tissue PK parameters are determined.Example: Definitive Tissue Distribution & PKMean - µg equiv/g tissueTissue Type Tissue 1 h 3 h 6 h 12 h 24 h 48 h 72 h 168 h 696 hVascular/ 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 BQLCentral 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 BQLEndocrine 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 BQLFatty 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 BQLDermal 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 BQLReproductive 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 BQLSkeletal/Muscular Bone 0.191 0.136 0.749 0.494 0.093 0.036 BQL BQL BQLSkeletal/Muscular Heart (myocardium) 14.338 16.461 11.706 8.661 6.419 0.624 0.037 BQL BQLSkeletal/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 BQLOcular 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
  14. 14. Autoradiographs showingthe tissue distribution inalbino (Sprague-Dawley)and pigmented rats (LongEvans).Note the amount ofradioactivity in the eye ofthe Long Evans rat vs. theSprague-Dawley rat.Data is routinely used todetermine HumanRadiation Dosimetry in>40 tissues.Example: Definitive TD and Tissue PK
  15. 15. 5/17/2013 15Example: Radiation DosimetryHuman radiolabeled drug studies are performed as part of Phase II clinicaltrials to determine human metabolism and pharmacokinetics of new drugentities. 14C-and 3H-labeled compounds are routinely usedDosimetry predictions rely on mathematical models and radioactivetissue/organ concentration and/or excretion data, which are obtained fromradioactive dosing animal studies (typically, rodents).Various methods to determine human and dosimetry predictions havebeen published by FDA and the International Commission on RadiationProtection (ICRP), but the calculations and data obtained from the variousmethods can produce different predictions.Different Pharma companies and CROs have developed different methodsover the years and some being used today are outdated and/orinappropriate when using QWBA data and 14C or 3H.
  16. 16. 5/17/2013 16Hendee-Marinelli MIRD ICRP w/out W.F.D (rem) Dman (rem) mSV remAdipose (brown) 0.02554 0.21449 0.09320 0.00932Adipose (white) 0.00845 0.16601 0.07213 0.00721Adrenal Gland 0.08143 0.28389 0.12335 0.01234Blood (cardiac) 0.01904 0.06137 0.02667 0.00267Bone (femur) 0.00576 0.17537 0.07620 0.00762Bone Marrow (femur) 0.02236 0.09351 0.04063 0.00406Brain 0.00259 0.06318 0.02745 0.00275Cecum 0.02854 0.38506 0.16731 0.01673Epididymis 0.05857 0.22230 0.09659 0.00966Eye Lens 0.00585 0.04834 0.02100 0.00210Eye Uveal Tract 0.02214 0.35203 0.15296 0.01530Heart 0.07229 0.19932 0.08660 0.00866Large Intestine 0.11983 0.59201 0.25724 0.02572Liver 0.18987 0.17898 0.07777 0.00778Lung 7.17978 20.63142 8.96459 0.89646Lymph Node 0.03693 0.19621 0.08525 0.00853Pancreas 0.05170 0.19386 0.08423 0.00842Pituitary Gland 0.06679 0.20283 0.08813 0.00881Prostate Gland 0.03461 0.24700 0.10733 0.01073Renal Cortex 0.42589 0.54124 0.23518 0.02352Renal Medulla 0.17270 0.43635 0.18960 0.01896Salivary Gland 0.02316 0.10613 0.04612 0.00461Seminal Vesicles 0.02526 0.19243 0.08361 0.00836Skeletal Muscle 0.02067 0.20226 0.08788 0.00879Skin 0.02868 0.25401 0.11037 0.01104Small Intestine 0.02063 0.30142 0.13097 0.01310Spinal Cord 0.01892 0.31804 0.13819 0.01382Spleen 0.11811 0.26489 0.11510 0.01151Stomach (gastricmucosa) 3.17550 6.73324 2.92567 0.29257Testis 0.00789 0.06870 0.02985 0.00298Thymus 0.00970 0.07178 0.03119 0.00312Thyroid 0.02839 0.21398 0.09298 0.00930Urinary Bladder 0.38319 0.73106 0.31765 0.03177Whole Body Total 12.49077 0.72000 1.52825 0.15282Example: Radiation DosimetryConclusions of a Comparison:Different calculations canproduce different predictions ofradiation exposure.Some calculations (i.e. GItransit model), which aredeveloped for penetratingradiation (e.g., PET, Spect,Gamma Scintigraphy) are notappropriate for predicting 14Cand 3H exposures and can overestimate actual tissueexposure.
  17. 17. Examples – Tumor PenetrationEnables the distinction between the necrotic and solid portions ofa tumor that can have very different concentrations.Provides a way to see if there are other potential therapeutictargets for the compound by determining concentrations in othertissues.
  18. 18. Examples – Ocular Drug DistributionPhosphor imaging and MARG can be used toexamine quantitative distribution of radiolabeledcompounds in fine ocular structures of rats, rabbits,and dogs.
  19. 19. Example: Routes of EliminationAutoradiograph of bile-ductcannulated rats given an IVdose of a 14C-labeled drugQWBA demonstratedintestinal secretion as anunanticipated route ofelimination
  20. 20. Adult and Fetal Brain Distribution andMetabolism of 14C-AZTBackground and Study Design17 May 2013 Confidential 20Proof of principal study on Placental Transfer to demonstrate the utility of QWBA forthis examination.Combination study to examine fetal and maternal tissue distribution of14C-azidothymidine (14C-AZT) after a single intravenous administration to a pregnantfemale rat.QWBA revealed differential distribution of 14C-AZT-derived radioactivity in fetal andmaternal, brain and liver. Concentrations of radioactivity in fetal brain and liver werehigher than in the adult.Fetal and maternal brain and liver were obtained by necropsy of an additionalpregnant rat for MARG and metabolite profiling by radio-HPLC.To further characterize the different patterns of distribution, samples of fetal andmaternal brain and liver were homogenized, extracted and analyzed by radio-HPLC toobtain a metabolite profile of each tissue and differences were identified.Further analysis using mass spectroscopy techniques provided identification of thesemetabolites.
  21. 21. Adult and Fetal Brain Distribution andMetabolism of 14C-AZTQWBA ResultsWhole-body Autoradioluminographs of an pregnant rat (day17) (left) and a 17-day old fetus (right) showing differentialdistribution 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. 22. Adult and Fetal Brain Distribution andMetabolism of 14C-AZTMicro-Autoradiography in Brain and LiverPhotomicroautoradiographs of the cellular localization of 14C-AZT-derivedradioactivity 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 22Maternal TissueFetal TissueBrain Liver
  23. 23. Adult and Fetal Brain Distribution andMetabolism of 14C-AZTAZT MetabolismRadiochromatographs showing the metabolite profiles obtained frommaternal and fetal liver and brain samples after a single intravenousadministration of 14C-AZT.17 May 2013 Confidential 23Liver:Brain:
  24. 24. Brain Distribution and Efficacy of a 14C-siRNA forHuntington’s DiseaseBackground and Study DesignHuntington’s disease is caused by a n overexpression of the CAG repeatin the Huntington gene (Htt). Normally, this section of DNA is repeated 10to 28 times. But in persons with Huntingtons disease, it is repeated 36 to120 times.The Sponsor of this study worked with QPS to study the distribution andefficacy of an administered 14C-siRNA in rats.14C-siRNA was directly infused into the striatum of rat brains over timeperiods up to 7 days.Each brain was removed at different time points after dosing and werefrozen and sectioned for quantitative autoradiography analysis andanalysis of tissue punches by real-time PCR.17 May 2013 Confidential 24
  25. 25. Brain Distribution and Efficacy of a 14C-siRNADosing and Sample CollectionSprague Dawley Rats were fitted with indwelling canulas that werestereotaxically positioned into the striatum of the brain.14C-siRNA was infused into the striatum at slow rates over times up to 7days.The brain was removed and flask frozen on dry ice, and plasma wascollected.17 May 2013 Confidential 25
  26. 26. Brain Distribution and Efficacy of a 14C-siRNAQuantitative Autoradioluminography in BrainFrozen Brains were cryosectionedthrough the striatal region at 40 µmthickness, sections were collected ontoglass slides, and immediately dried on aslide warmer.Brain sections were exposed tophosphor imaging plates along with 14Ccalibration standards for 4 days and theimaging plates were scanned at aresolution of 50 µm.Brain concentrations were determinedat discreet locations throughout thebrain to create a detailed histogram ofconcentrations through the injectionsite.17 May 2013 Confidential 26
  27. 27. Brain Distribution and Efficacy of a 14C-siRNAQuantitative Autoradioluminography in Brain17 May 2013 Confidential 27
  28. 28. Brain Distribution and Efficacy of a 14C-siRNAQuantitative Real-Time PCR in BrainBrain Punch samples were collected from various regions in sections thatwere adjacent to those collected for autoradiography duringcryosectioningSamples were analyzed by rtPCRResults showed that the Htt gene was silenced.17 May 2013 Confidential 28Sample Detector Ct ∆ CtAvg∆Ct∆CtSD∆Ct%CVRat #1_300u_2 GAPDH 26.46927.07027.6658 0.7816 10.1961Rat #1_300u_2 Htt 33.5393Rat #1_300u_2 GAPDH 26.41088.5509Rat #1_300u_2 Htt 34.9616Rat #1_300u_2 GAPDH 26.54147.3764Rat #1_300u_2 Htt 33.9178
  29. 29. Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelBackgroundNiemann-Pick Disease Type C is caused by an accumulation of materials(cholesterol and other fatty acids) in the bodys cells that leads toprogressive intellectual decline, loss of motor skills, seizures anddementia. The disease progresses at varying rates. Young children whodisplay neurological symptoms generally have an aggressive form of thedisease, while others may not display symptoms for years.The Sponsors of this study (Jansen R&D, LLC, and University ofPennsylvania) worked with QPS to study the distribution of anadministered 14C-Cyclodextrin in a Feline Niemann Pick Model tocharacterize the spatial distribution and pharmacokinetics in the centralnervous system and other organs to gain a better understanding for thetreatment of the disease in Humans.17 May 2013 Confidential 29
  30. 30. Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelStudy DesignFemale Niemann-Pick Cats (Univ. of Penn) wereadministered a single intrathecal dose of 14C-Cyclodextrin at120 mg/cat (200 µCi/cat)One cat per time point was euthanized at 0.25 h, 1 h, 4 h, 8h, 12 h, and 24 h post-dose, and each carcass was frozen forQWBA analysis.Concentrations of Cyclodextrin were determined in > 30tissues including discreet regions of the brain.17 May 2013 Confidential 30
  31. 31. Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelResults17 May 2013 Confidential 318 h 12 h 24 h
  32. 32. Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelResultsTissue PK Parameters of Cyclodextrin (µg/g tissue) in Niemann-Pick Cats17 May 2013 Confidential 32Tissue AUCall AUCinf_obs Cmax Tmax T1/2# of pt.s inT1/2 r2ug equiv·h/g ug equiv·h/g ug equiv/g h hAdrenal Gland 205.470 250.919 32.709 0.25 10.097 3 0.93Blood (cardiac) 140.164 159.866 40.011 1 10.8 4 0.17Brain (cerebellum) (hi) 3620.677 4072.258 689.516 1 6.3 5 0.53Brain (cerebellum) (low) 405.069 Missing 22.485 24 Missing 0 MissingBrain (cerebrum) (hi) 3184.710 5686.564 417.944 4 32.6 3 0.78Brain (cerebrum) (low) 365.502 621.691 25.759 12 13.0 2 1.00Brain (medulla) 969.982 1089.337 116.422 4 6.8 2 1.00Kidney Cortex 484.666 Missing 33.101 24 Missing 0 MissingLiver 65.103 106.111 12.267 1 ND 5 0.22Nasal Turbinates 3163.073 3952.117 1031.483 0.25 17.555 3 0.90Pituitary Gland 2278.187 2406.100 426.351 0.25 ND 4 0.61Skeletal Muscle 16.341 21.900 2.791 1 ND 3 0.60Spinal Cord 1782.007 2105.836 214.885 4 8.6 4 0.57Urinary Bladder 107.586 235.612 15.243 1 ND 4 0.62ND = Not Determined due to insufficient data
  33. 33. Further Image analysisprovided a histogram ofBrain concentrationsfrom which data wasextracted to obtain PKparameters at discreetlocations throughoutthe Brain17 May 2013 Confidential 33Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelResults
  34. 34. Distribution of 14C-Cyclodextrin in FelineNeiman-Pick C ModelConclusionsDrug-derived radioactivity was absorbed from the cerebellomedulary cistern and waswidely 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 andmost other tissues were decreasing, penetration of drug-derived radioactivity into thedeeper parts of the CNS tissues was ongoing and concentrations in different regions of thebrain varied over 24 h.Tissues, besides the CNS, with the highest concentrations (≥ 100 µg equiv/g) ofradioactivity 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 routeof elimination.Prolonged exposure of tissues that are outside of the CNS is expected, albeit at lowconcentrations, as drug-derived radioactivity is eliminated from the CNS compartment andthen eliminated from the body.17 May 2013 Confidential 34
  35. 35. Overall ConclusionsQWBA provides detailed, discreet, quantitative, tissuedistribution and detailed PK information for small and largemolecule drugs.MARG provides detailed, discreet, qualitative, cellulardistribution information for small and large molecule drugs.Several other analytical techniques such as LC/MS/MS, andrtPCR can be easily combined to provide a wealth ofknowledge regarding the detailed distribution, concentrationand kinetics of various test drugs in the central and peripheralnervous system of laboratory animals.17 May 2013 Confidential 35
  36. 36. AcknowledgementsQPSAlfred LordiPaul StrzemienskiTony SrnkaJackie MorganJackie EckboldSarah PattersonYvette WarnerMartin HulseMarna DiOssiHelen ShenZamas LamBen Chien17 May 2013 Confidential 36Charles Vite, DVM, Ph.D.Janssen Research& Development, L.L.C.Universityof PennsylvaniaMark Kao, Ph.D.

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