2005 SWBA Presentation Abstracts
Tissue Distribution of a Novel Antibacterial Agent
A. Lordi 1, W. Waddell 2, E. Solon 1, B. Chien 1, 1Quest Pharmaceutical Services, Newark DE, USA, 2University of Louisville, Louisville Kentucky
This study investigated tissue distribution, and tissue concentration of a novel antibiotic agent and its metabolites using quantitative whole-body autoradioluminography. Twelve rats were given a single intravenous administration at a dose of 3 mg/kg ( ≫ 184 m Ci/kg). Upon image analysis, an interesting finding was observed. “Foci” of a high concentration of radioactive material were observed in some tissues of rats at all time points. These foci were not fully consistent with the appearance of radiation distribution that results from precipitation of test materials in small capillaries of certain organs such as lung and kidney, where the test material has poor solubility. An additional group of four rats were given a single oral administration at a dose of 3 mg/kg ( ≫ 161 μCi/kg). The foci were not observed after oral dosing. The novel antibiotic is a bipolar compound, its structure suggests it can form several pi bonds aligned in such a way that can lead to non-covalent bonding to itself in a stacking pattern, similar to micelle. It is also possible that this novel antibiotic has poor solubility and the presence of the foci are atypical as compared to other poorly soluble materials and their precipitation. Blood and most tissue data showed concentrations were half as high in the oral dosed animals and this suggested the foci were an artifact of the dosing route and are probably concentration dependant.
Patterns of penetration of plaque imaging agents under study in rabbit aorta plaque model ex vivo
Eric J. Wexler, Simon Robinson, and Padmaja Yalamanchili
Bristol-Myers Squibb Medical Imaging 331 Treble Cove Road , North Billerica , MA 01862 , 978-671-8485
Imaging of atherosclerotic plaque using radiotracers requires an appropriate understanding of plaque uptake and localization of the imaging agent . An ex-vivo incubation model has been developed that allows the examination of some parameters needed for compound optimization. Plaque bearing tissue from the aorta of rabbits fed a hyperlipidemic diet 16 to 20 weeks post angioplasty was used to examine tissue penetration and tissue localization.
Four compounds (Diphenylhydantoin, Iodoantipyrine, Testosterone, and Caffeine) were selected to represent the range of logD from highly hydrophobic to highly hydrophilic. Plaque penetration was examined from the lumen side using a customized incubation chamber. The distance of penetration of 14C labeled compounds was studied at 15 minutes by autoradiography of tissue cryosections. The autoradiograph was image enhanced optimized for edge detection and overlaid onto photographic images of unstained tissue. Hematoxylin and Eosin, Trichrome, and Oil red O staining was performed to evaluate the atherosclerotic plaque components and regions. In a separate ex vivo study the localization of 14C Testosterone as determined by autoradiography was then correlated to these regions.
In the penetration assay hydrophilic compounds penetrated further into the plaque than hydrophobic compounds. Caffeine penetrated through the entire vessel, Iodoantipyrine and Testosterone penetrated to the media while Diphenylhydantoin just penetrated into the plaque cap. In the tissue localization assay 14C Testosterone showed a pattern of localization with areas of higher activity located in the plaque core.
The model developed has allowed the assessment of the characteristics needed for rapid plaque penetration and tissue localization. This evaluation may provide insight into the potential of compounds for atherosclerotic plaque imaging.
Radioluminography in minipigs to estimate in vivo melanin binding and dosimetry of radiolabeled compounds in humans
B. Hofmann , M. Friebe, P. Muschick, V. Uhlendorf, A. Lippoldt, C. Guenther, Schering AG, Berlin, Germany
Objective : The investigations were designed to estimate in vivo melanin binding in human eyes following intravenous administration of a I-131-labeled test compound with a potential of melanin binding. The use of minipigs as a representative model was evaluated.
Dosimetry calculations are a prerequisite for radioactive biotransformation and mass balance studies in humans. Several lipophilic compounds such as procainamide, chlorpromazine etc. are known to display significant eye uptake in humans, due to a strong melanin binding. This may lead to high radiation doses in the eye. Therefore, we have evaluated a minipig model to determine quantitative eye accumulation. Whole body distribution and elimination of the test compound were examined in minipigs using a Gamma-camera. In order to further determine the amount of I?131?radiolabel bound to melanin containing structures in minipig eyes, cryosections of excised eyes were quantitatively evaluated using radioluminography. In addition, histological sections of minipig and human eyes were incubated in vitro with the test compound and quantitatively evaluated using radioluminography in order to compare the extent of melanin binding of the radiolabel in the eyes of the respective species.
Methods : 9 mini pigs (Gottingen Minipigs) received between 4 and 5.3 MBq/kg I?131?labeled test compound intravenously. At defined time points until 3 weeks post dose animals were examined using a Gamma-camera to obtain time dependent planar and SPECT images for determination of whole body distribution and elimination of the test compound.
Following sacrifice at defined time points post dose (1 h, 6 h, 12 h, 1 d, 2 d, 3 d, 1 week, 2 weeks and 3 weeks) both eyes were excised in order to quantify the amount of I?131?radiolabel in toto using gamma-counting. From the right eye iris, ciliary epithelium, RPE (retinal pigmented epithelium) and choroid were separated and the concentration of I?131?radiolabel was determined by gamma counting as well. In the left eye the distribution of radiolabel was examined using quantitative radioluminography. Cryosections of the left eye were analyzed together with blood standards spiked with I?131 using the BAS 5000 system and the AIDA software ( Raytest , Germany ). Concentrations of the I-131-radiolabel were determined in ciliar body and choroid/RPE and elimination half-lives were determined.
In addition, to compare the extent of melanin binding in eyes of mini pigs and humans, respective histological sections were incubated with the I-131-labeled test compound in vitro. The amount of radiolabel bound to ciliar body and choroid/RPE was determined using radioluminography.
Results : The compound was rapidly eliminated from the body but a longer lasting retention in the eyes was observed. Radioluminography of excised mini pig eyes showed maximal radiolabel concentrations in choroid/RPE and ciliar body between 1 and 24 h post dose, thereafter concentrations declined. After 3 weeks a significant amount of radiolabel was still detectable. The terminal elimination half-life of I?131 radiolabel in ciliar body and choroid/RPE was between 150 and 200 h. Blood radiolabel concentrations decreased below the limit of detection at 24 h post dose. In vitro binding studies on human and mini pig eyes showed that the amount of radiolabel bound in melanin containing structures was similar. A dosimetry estimation for the human eye was performed based on the distribution obtained in the mini pig.
Combined Tissue Distribution, Tissue Quantitation, and Metabolite Profiling using Quantitative Whole Body Autoradiography (QWBA), Flow Scintillation Detector (FSD) and LC/MS/MS
E. Solon, A. Lordi, L. He, L. Venkataragan, Y-S. Chen, H. Shen, Z. Lam, Y. Zhu, R. Hamler, and B. Chien, Quest Pharmaceutical Services, Newark DE, 19711, USA
This study addressed tissue distribution, metabolic profile, metabolite identification, and tissue concentration of Paclitaxel (Taxol) and its metabolites by combining QWBA, FSD and LC/MS/MS in a single experimental protocol. Four rats were given a 1h IV infusion of Taxol at a dose of 48 mg/kg (40 m Ci/kg). One rat each was euthanized immediately at the end of infusion and at 4-h post infusion. A third rat died during dosing after receiving approximately ? of its dose, and a fourth rat was found dead at 17h after the end of infusion. All carcasses were flash-frozen, prepared for QWBA, and tissue sampling for FSD and LC/MS/MS. QWBA results provided tissue distribution information as total radioactivity. Liver, spleen, heart, lung, blood, bone marrow, muscle, kidney, urine, and feces, which had the highest concentrations of the radioactivity, were removed from the remaining frozen carcasses for further analyses. Metabolites were identified using LC/MS/MS coupled with FSD. Concentrations of the parent Taxol and its metabolites in each organ or sub-region were quantitated using LC-radiochromatography and QWBA. Quantitation of drug and metabolites were also accomplished using LC/MS/MS. This study demonstrated the advantage of QWBA as a powerful tool for elucidating the distribution of Taxol and its metabolites in all the tissues and sub-regions. Furthermore, the combination with FSD or LC/MS/MS, enabled the determination of parent and metabolite concentrations in specific target tissues. We are currently using this combined QWBA-LC/MS/MS-LC/FSD approach for both discovery and non-GLP studies.
MALDI IMAGING: A New Technique for Drug Distribution Studies
Julie Wingate, Applied BioSystems, MDS Sciex
Mass spectrometry is an extremely powerful technique in determining the molecular weight of a sample, especially when combined with techniques such as HPLC. When it comes to the analysis of tissue samples, however, LC-MS analysis provides information on the individual compounds in the tissue but all spatial relationship information on how those compounds are distributed throughout the tissue is lost. The information on the spatial distribution of drugs in tissues is extremely important in determining whether or not a drug is reaching its target site or accumulating elsewhere in the body. To address this, imaging of drugs directly from tissues using MALDI with hybrid quadrupole time-of-flight (QqTOF) has started gaining more attention recently. A key strength of combining a QqTOF with MALDI is the ability to perform product ion scans, which can improve signal-to-noise dramatically and also assists with compound identification.
One of the most exciting applications of MALDI Imaging is the study of drug and metabolite distribution in tissue samples. Unlike other traditional techniques for drug distribution studies, MALDI imaging can differentiate between drugs and metabolites and can produce accurate mass data, allowing for conclusive structural identification.
Applied Biosystems/MDS Sciex has developed a novel MALDI imaging software package to acquire and process data from the QSTAR O System. This presentation will provide detailed information on the instrument and imaging features and illustrate the application of the technique to the study of drugs and metabolites in liver tissue.
Utilizing Imaging Mass Spectrometry for Drug Distribution Studies
J Chen, Y Hsieh, W Korfmacher , L Crossman, N McHugh , I Knemeyer
Schering-Plough Research Institute, Kenilworth , NJ 07033
Drug distribution studies are often critical in understanding the pharmacological action of new chemical entities, yet they are rarely performed in the drug discovery stage due to the lack of radio-labeled compounds and the limited resources from drug disposition groups. In this work, we use MALDI mass spectrometry to generate two dimensional ion density maps in animal tissue sections. Advantages of this emerging technology include versatility (no need for radio-labeled compound) and specificity (signal is generated from the parent drug molecule only, not the drug-derived material). We will compare results on the same tissue section using both imaging mass spectrometry autoradiography. In addition, examples will be given to illustrate how it can be used to answer distribution questions in drug discovery. Current limitations for this technique will also be discussed.
Improvement of placental transfer methodology
Application in rabbit
Eric ERDOCIAIN, Magali MARTIN-CHAVE(*) and Patrick DUPONT
Sanofi Aventis, Research center, 195 route d'Espagne, F-31036 Toulouse.
(*)Trainee in the GMPK department
Placental transfer studies in rabbit are designed to support developmental toxicological studies in this species, especially when results of these studies are negative. However, the study design is not clearly defined. Dose to be used, single or repeated administration and gestation day and dosing day are recurrent questions for which answers are needed.
Our current rabbit placental transfer model exhibited drawbacks which were a limitation for a deployment within the company.
In order to overcome study design, a review of the litterature was conducted in parallel of the validation of a new model for placental transfer by caesarizing fetuses directly from euthanasiaed rabbit pregnant females.
Model was firstly improved without administration of compound ( day of caesarian, sampling of fetuses ,freezing methodology ...) then was cross-validated against the "current model" using three radioactive compounds ( two known to cross the hematoplacental barrier and one exhibiting no passage)
Review of the litterature, results of the validation and strategy will be presented and discussed.
Localization of Molecules Tagged With Stable Isotopes: Multiple Imaging Mass Spectrometry at the Tissue to Sub-cellular Levels
Andrew Davis, Cameca Instruments, Inc.
(ABSTRACT NOT AVAILABLE)
The Photon Imager : a new way of performing
Bioluminescence/Biofluorescence in-vivo
Etienne Durriseau, Biospace Mesures
The photon Imager is a new generation of in-vivo Bioluminescence/Biofluorescence imager, which unlike its existing competitors, uses an intensified CCD camera instead of a cooled down CCD camera.
Indeed, the main drawback of cooled down CCD camera based instruments performing in vivo imaging of luciferin/luciferase (bioluminescence) or of GFP, ds Red or other fluorophors (biofluorescence), is the irreducible constant read out noise that prevents fact acquisitions, hence forbidding true real time, kinetic determinations.
The Photon Imager is based on our experience of intensified CCD camera's that we have extensively be using in our digital, real time autoradiography instruments : the Beta Imager and the Micro Imager that most of the people attending this meeting know since a long time.
With the Photon Imager we are using a third generation intensifying tube that allows to count photons emitted by a living animal (mouse or rat) in the range 400 to 900 nm as a result of a luciferin/luciferase reaction in genetically modified animals or fluorescence of (e.g.) Green Fluorescent Protein injected into an animal.
Examples of such types of imaging will be shown as well as potential applications using very fast acquisitions and phosphorescence of nanoparticles specific of antibodies, drugs, etc...
This type of new imaging modality is becoming very popular in pharmaceutical research whether in academic or pharmaceutical companies environment.
GLP conform documentation by using the Leica CM3600 instrument software
Claudia Dorenkamp, Product Manager Sectioning and Cryosectioning; Leica Microsystems Nussloch GmbH, Heidelberger Strasse 17-19, D- 69226 , Nussloch , Germany
The Leica CM3600 instrument software allows creating and recording GLP conform documentation of individually tailored studies including all events and alarms occuring during the duration of the study.
Documentation of either all or only selected sections with a choice of parameters, such as set and actual section thickness, temperature and knifeholder position can be created.
To facilitate the link between the documentation and the section ? a label with any required information can be printed and attached to the section.
A password and user identification is required to open and close the system. The system administrator ensures that only approved users have access to various levels within the operating system.
The precise photo position allows complete photo documentation of all desired levels of the cryoplaned block (even 3 dimensional reconstructions).
AIDA: New Software Tools to Quantify WBA Images
Mike Treuner, Raytest
For many years the imaging plate technology has been used successfully to quantify tissue sections in Whole Body Autoradiography (WBA).
According to the regulations for storing and retrieving electronic records, the integrity of raw and evaluation data is essential in any data analysis. Raytest’s WBA software AIDA guarantees the integrity of all relevant data used for the evaluation and protects them from intentional or unintentional modification.
Integration / quantification of tissue sections that contain very low amounts of radioactivity has always been a difficult procedure. The new version of AIDA includes software tools to simplify this process. The radioluminography image of a tissue and its positive image (obtained by a flatbed scanner) are overlaid to quantify low-activity sections easily and precisely.
Innovation in Vibratome WBA Cryo-Microtome.
Drew Mehta, Vibratome Company
Since the introduction of the modern technique of Whole Body Autoradiography (WBA) by Sven Ullberg in 1954, the WBA technique has advanced in many different fields. There have also been significant advancements made with imaging techniques. Some of these new imaging techniques, such as electronic autoradiograms (Beta-Imager) and phosphor imaging plates, have demonstrated their significance compared to the original technique of film exposure. It is also important to note that histological methods are being refined which allows the field of autoradiography (Drug Metabolism) to improve the analysis of the sections.
With the inception of WBA-cryomicrotome, initially with Bright Instruments and recently with Leica Microsystems, the days of working in cold freezers are a distant past. Although the advances made in other fields of WBA have been significant, the cryomicrotome has gone through little changes in recent years. In keeping with the tradition of efficiency, automation, accuracy and high-throughput, the Vibratome Company has designed the most advanced cryomicrotome to suite today’s big-pharma and CRO needs.
With its Smart Tag? tracking system, researchers will be able to track the study subjects and corresponding sections from the process of selection to analysis. The automated dehydration system will allow researchers to continually section study subject blocks whilst the previous individual sections/frames are automatically transferred to a dedicated dehydration chamber. This dehydration chamber will track each section while the software will record vital information such as temperature, section thickness, section number, dehydration conditions and storage information. This dedicated dehydration chamber will allow the researcher to continuously use the cryomicrotome for sectioning, hence establishing higher productivity and efficiency. The software will also have an interface for automated bulletin board reporting for critical reports. The new cryomicrotome will have an option for the macrotape transfer system (MTTS ? Instrumedics) to be installed internally. This will allow the researcher to transfer a section for histological staining with more ease. The Vibratome Company is also currently working on an imaging system, which will allow you to take 3-D episcopic images from the tissue block surface.
It is the hope of The Vibratome Company that with these innovations, valuable time is better spent performing analysis of the researchrather than sectioning, dehydration, imaging and data processing.
The Issues of [ 3H] Quantification in QWBA
Alain SCHWEITZER, PhD, Gian CAMENISCH, PhD & Gerhard GROSS, PhD, Novartis Pharma AG, CH-Basle
The quantitative whole-body autoradiography “blood scale” method is based, among others, on a similar absorption of the β ? radiations in the major tissues, as investigated and demonstrated by the speaker for 14C-labeled material [1,2]. This method has been used so far successfully for more than 10 years by several dozens of autoradiographers worldwide, and has been shown to allow for the accurate estimation of tissue radioactivity concentrations in whole-body sections.
However a lot of questions and uncertainties have been raised recently, in particular whether blood may be realistically used to quantify 3H-labeled radioactivity in all the tissues.
Therefore studies were conducted at Novartis Pharma to assess the radioactivity concentrations in typical blood, brain and liver sections using different 3H-labeled blood-, liver- and brain- homogenates of known radioactivity concentrations as calibrators. Selected sections were processed for autoradioluminography (exposure onto imaging plates) and for direct nuclear counting (exposure into a BetaImager 2000 counter); the PSL- and nuclear events- derived radioactivity concentrations were compared to the “theoretical” ones as estimated by liquid scintillation counting, as well as to those obtained using the “classical” blood scale method.
The results will be discussed from both a theoretical and a practical point of view.
References:
[1] Schweitzer A, 1984. Autoradiographie qualitative et quantitative. Application a une substance dopaminergique (CU 32-085) et a l’un de ses metabolites principaux (CH 29-717) chez le rat. PhD thesis, Universite de Nancy, France.
[2] Schweitzer A et al., 1987. A simple method for the quantitation of [ 14C]whole-body autoradio-grams. Int J Appl Radiat Isot; 38(5):329?333.
Quantification of 3H-Labeled Compounds for Drug Discovery QWBA Studies
Mike Potchoiba, Pfizer, Groton , CT , USA
Whole?body autoradioluminography (WBAL) has evolved as the preferred method for conducting tissue distribution studies. WBAL use in drug discovery is not as prominent as it could be which is probably due to the lack of available radiolabeled compounds. The ability to use tritium as the radiolabel is of key importance as this material can in general be synthesized more readily than carbon?labeled drug candidates. Since the preparation of test material is a major, gating?factor in study turnaround, the use of tritium is key to the timely completion of experiments in the discovery setting. A series of experiments were designed to assess WBAL utility using tritium for drug discovery as early as Lead Development. The objective of one experiment was to compare the tissue distribution of [ 3H]glucose with the distribution pattern of [U? 14C]glucose. Additional studies were conducted to identify a tissue compartment responsible for observed triphasic pharmacokinetics, characterize the ability of a drug entity (DE) to cross the blood?brain barrier, and compare tissue pharmacokinetics between two rat strains. Tritium and carbon?14 glucose tissue distribution patterns were similar demonstrating that tritium can be substituted for carbon?14 which is of crucial importance in drug discovery since tritium synthesized DE are more readily obtainable than [ 14C]DE. The next series of experiments illustrated: a) identification of adipose as the tissue compartment responsible for an extended terminal elimination phase, b) sustained penetration of a DE into CNS tissues for 24 hr, and c) tissue distribution differences between two DE of the same therapeutic class. The results of these studies demonstrated that WBAL could be successfully used to characterize the distribution of tritium-labeled candidates in drug discovery. In any analytical method, the calibration curve needs to be constructed with standard curve calibrators (STD) over a concentration range expected in the samples being analyzed. The inclusion of cryosection quality control samples (CQCS) provides a quantitative measurement of sensitivity and the foundation for determining the error associated with the WBAL methodology. Calculating the accuracy and precision of quality control samples assesses the quality of any analytical method. In these WBAL experiments, CQCS were used to monitor the quality of the WBAL analytical method. The use of CQCS ensured that any variation in obtaining cryosections of the specimens would also be reflected in the phosphor imager signal produced by the CQCS. Variations in section thickness would produce similar variations in the intensity of the phosphor imager signal. The greater the variation in section thickness, the greater the deviation will be from the expected nominal concentration of radioactivity. Thus, the inclusion of CQCS in the same block as the specimens was an appropriate measure of accuracy and precision for testing the validity and quality of this WBAL analytical method.
The results of these experiments illustrated that a good linear correlation exists between the concentration of tritium radioactivity and the phosphor imager response. In comparison, the STORM phosphor imager has a lower limit of quantification of 1.6 nCi/g for carbon?14 and has a linear dynamic range that expands across 5 orders of magnitude (i.e. 1.6 to 39,000 nCi/g). Based upon the lower quantification limits, WBAL analysis employing tritium appears to be approximately 50?fold less sensitive than that observed for carbon?14.
These WBAL experiments demonstrated the value of this methodology as a screening tool to assist with the selection of a drug candidate. WBAL utilization in drug discovery provides insightful data towards designing pharmacology and toxicology experiments.
MillitroN to ZenoN software interface
Patrick DUPONT 1 , Bruno Hugon 2, 1 Sanofi~Synthelabo Recherche, 195 route d'Espagne, F-31036 Toulouse Cedex, 2 Leica Microsystemes SAS, 86,Avenue du 18 Juin 1940, F- 92563 Rueil-Malmaison
In order to improve quality environment of cryosectioning, a Millitron to Zenon software interface, based on the measurement of the reproducibility of the knife holder displacement during sectionning has been developed in collaboration between MPK Department of Sanofi-Aventis, Research Center of Toulouse, and Leica Microsystems.
An external micro-incremental probe is connected to the knife holder of a microtome. The values of the displacement of the knife holder are collected for each section and transmitted to a Millitron device. Each time the foot switch is activated, the value displayed on the screen of the Millitron device is updated in the record file of the Zenon software in the same way that other parameters selected.
The external micro-incremental probe and the Millitron device are manufactured by Mahr GmbH, Esslingen , Germany .
Zenon software has been developed for Leica-Microsystems GmbH, Nussloch , Germany .
This interface is full validated and used in routine in GMPK Department of Sanofi-Aventis in Toulouse .
The presentation will be focused on the description and on the functionnalities of Millitron to Zenon software interface.