Name: rapid evaluation of toxicity of chemical compounds using zebrafish embryos
Uploaded: 2019-08-25T14:00:00
Description: Watch this Scientific Journal Video about Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos at JoVE.com

The work was supported by grants from Sigrid Juselius Foundation (SP, MP), Finnish Cultural Foundation (AA, MH), Academy of Finland (SP, MP), Orion Farmos Foundation (MH), Tampere Tuberculosis Foundation (SP, MH and MP) and Jane and Aatos Erkko Foundation (SP and MP). We thank our Italian and French collaborators, Prof. Supuran, and Prof. Winum, for providing carbonic anhydrase inhibitors for safety and toxicity evaluation for anti-TB and anti-cancer drug development purposes. We thank Aulikki Lehmus and Marianne Kuuslahti for the technical assistance. We also thank Leena M&#228;kinen and Hannaleena Piippo for their help with the zebrafish breeding and collection of embryos. We sincerely thank Harlan Barker for critical evaluation of the manuscript and insightful comments.

The zebrafish core facility at Tampere University has an establishment authorization granted by the National Animal Experiment Board (ESAVI/7975/04.10.05/2016). All the experiments using zebrafish embryos were performed according to the Provincial Government of Eastern Finland, Social and Health Department of Tampere Regional Service Unit protocol # LSLH-2007-7254/Ym-23.
1. Setting Up of Overnight Zebrafish Mating Tanks
<ol>
	<li>Place 2-5 adult male zebrafish and 3-5 adult female zebrafish ...

<ol>
	<li>Amaouche, N., Casaert Salome, H., Collignon, O., Santos, M. R., Ziogas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marketing+authorization+applications+submitted+to+the+European+Medicines+Agency+by+small+and+medium-sized+enterprises:+an+analysis+of+major+objections+and+their+impact+on+outcomes.">Marketing authorization applications submitted to the European Medicines Agency by small and medium-sized enterprises: an analysis of major objections and their impact on outcomes.</a> Drug Discovery Today. 23 (10), 1801-1805 (2018).</li><li>Garg, R. C., Bracken, W. M., Hoberman, A. M., Gupta, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+and+developmental+safety+evaluation+of+new+pharmaceutical+compounds.">Reproductive and developmental safety evaluation of new pharmaceutical compounds.</a> Reproductive and Developmental Toxicology. , 89-109 (2011).</li><li>Lee, H. Y., Inselman, A. L., Kanungo, J., Hansen, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+models+in+developmental+toxicology.">Alternative models in developmental toxicology.</a> Systems Biology in Reproductive Medicine. 58 (1), 10-22 (2012).</li><li>Gao, G., Chen, L., Huang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-cancer+drug+discovery:+update+and+comparisons+in+yeast,+Drosophila,+and+zebrafish.">Anti-cancer drug discovery: update and comparisons in yeast, Drosophila, and zebrafish.</a> Current Molecular Pharmacology. 7 (1), 44-51 (2014).</li><li>Brown, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+test+chemicals+for+the+ECVAM+international+validation+study+on+in+vitro+embryotoxicity+tests.+European+Centre+for+the+Validation+of+Alternative+Methods.">Selection of test chemicals for the ECVAM international validation study on in vitro embryotoxicity tests. European Centre for the Validation of Alternative Methods.</a> Alternatives to Laboratory Animals. 30 (2), 177-198 (2002).</li><li>Selderslaghs, I. W., Van Rompay, A. R., De Coen, W., Witters, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+screening+assay+to+identify+teratogenic+and+embryotoxic+chemicals+using+the+zebrafish+embryo.">Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo.</a> Reproductive Toxicology. 28 (3), 308-320 (2009).</li><li>Brannen, K. C., Panzica-Kelly, J. M., Danberry, T. L., Augustine-Rauch, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+zebrafish+embryo+teratogenicity+assay+and+quantitative+prediction+model.">Development of a zebrafish embryo teratogenicity assay and quantitative prediction model.</a> Birth Defects Research Part B Developmental and Reproductive Toxicology. 89 (1), 66-77 (2010).</li><li>Hermsen, S. A., van den Brandhof, E. J., van der Ven, L. T., Piersma, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+embryotoxicity+of+two+classes+of+chemicals+in+a+modified+zebrafish+embryotoxicity+test+and+comparison+with+their+in+vivo+potencies.">Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies.</a> Toxicology in Vitro. 25 (3), 745-753 (2011).</li><li>Lessman, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developing+zebrafish+(Danio+rerio):+a+vertebrate+model+for+high-throughput+screening+of+chemical+libraries.">The developing zebrafish (Danio rerio): a vertebrate model for high-throughput screening of chemical libraries.</a> Birth Defects Research. Part C, Embryo Today: Reviews. 93 (3), 268-280 (2011).</li><li>Lantz-McPeak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+assay+using+high+content+screening+of+zebrafish+embryos.">Developmental toxicity assay using high content screening of zebrafish embryos.</a> Journal of Applied Toxicology. 35 (3), 261-272 (2015).</li><li>Truong, L., Harper, S. L., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+embryotoxicity+using+the+zebrafish+model.">Evaluation of embryotoxicity using the zebrafish model.</a> Methods in Molecular Biology. 691, 271-279 (2011).</li><li>Rodrigues, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+synthesis,+and+evaluation+of+hydroxamic+acid+derivatives+as+promising+agents+for+the+management+of+Chagas+disease.">Design, synthesis, and evaluation of hydroxamic acid derivatives as promising agents for the management of Chagas disease.</a> Journal of Medicinal Chemistry. 57 (2), 298-308 (2014).</li><li>Kanungo, J., Cuevas, E., Ali, S. F., Paule, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+model+in+drug+safety+assessment.">Zebrafish model in drug safety assessment.</a> Current Pharmaceutical Design. 20 (34), 5416-5429 (2014).</li><li>Peterson, R. T., Link, B. A., Dowling, J. E., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+developmental+screens+reveal+the+logic+and+timing+of+vertebrate+development.">Small molecule developmental screens reveal the logic and timing of vertebrate development.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (24), 12965-12969 (2000).</li><li>Stainier, D. Y., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+system+to+study+cardiovascular+development.">The zebrafish as a model system to study cardiovascular development.</a> Trends in Cardiovascular Medicine. 4 (5), 207-212 (1994).</li><li>Aspatwar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroimidazole-based+inhibitors+DTP338+and+DTP348+are+safe+for+zebrafish+embryos+and+efficiently+inhibit+the+activity+of+human+CA+IX+in+Xenopus+oocytes.">Nitroimidazole-based inhibitors DTP338 and DTP348 are safe for zebrafish embryos and efficiently inhibit the activity of human CA IX in Xenopus oocytes.</a> Journal of Enzyme Inhibition and Medicinal Chemistry. 33 (1), 1064-1073 (2018).</li><li>Rami, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-targeting+carbonic+anhydrase+IX+inhibitors+by+a+new+series+of+nitroimidazole-sulfonamides/sulfamides/sulfamates.">Hypoxia-targeting carbonic anhydrase IX inhibitors by a new series of nitroimidazole-sulfonamides/sulfamides/sulfamates.</a> Journal of Medicinal Chemistry. 56 (21), 8512-8520 (2013).</li><li>Spitsbergen, J. M., Kent, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+the+art+of+the+zebrafish+model+for+toxicology+and+toxicologic+pathology+research--advantages+and+current+limitations.">The state of the art of the zebrafish model for toxicology and toxicologic pathology research--advantages and current limitations.</a> Toxicologic Pathology. , 62-87 (2003).</li><li>Teraoka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cytochrome+P450+1A+is+required+for+circulation+failure+and+edema+by+2,3,7,8-tetrachlorodibenzo-p-dioxin+in+zebrafish.">Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish.</a> Biochemical and Biophysical Research Communications. 304 (2), 223-228 (2003).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nature Reviews Drug Discovery. 4 (1), 35-44 (2005).</li><li>Hill, A. J., Teraoka, H., Heideman, W., Peterson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+vertebrate+for+investigating+chemical+toxicity.">Zebrafish as a model vertebrate for investigating chemical toxicity.</a> Toxicological Sciences. 86 (1), 6-19 (2005).</li><li>Kari, G., Rodeck, U., Dicker, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+an+emerging+model+system+for+human+disease+and+drug+discovery.">Zebrafish: an emerging model system for human disease and drug discovery.</a> Clinical Pharmacology and Therapeutics. 82 (1), 70-80 (2007).</li><li>Gourmelon, A., Delrue, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+in+Support+of+Internationally+Harmonised+OECD+Test+Guidelines+for+Assessing+the+Safety+of+Chemicals.">Validation in Support of Internationally Harmonised OECD Test Guidelines for Assessing the Safety of Chemicals.</a> Advances in Experimental Medicine and Biology. 856, 9-32 (2016).</li><li>Aspatwar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-CA-specific+inhibitor+dithiocarbamate+Fc14-584B:+a+novel+antimycobacterial+agent+with+potential+to+treat+drug-resistant+tuberculosis.">beta-CA-specific inhibitor dithiocarbamate Fc14-584B: a novel antimycobacterial agent with potential to treat drug-resistant tuberculosis.</a> Journal of Enzyme Inhibition and Medicinal Chemistry. 32 (1), 832-840 (2017).</li><li>Kazokaite, J., Aspatwar, A., Kairys, V., Parkkila, S., Matulis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorinated+benzenesulfonamide+anticancer+inhibitors+of+carbonic+anhydrase+IX+exhibit+lower+toxic+effects+on+zebrafish+embryonic+development+than+ethoxzolamide.">Fluorinated benzenesulfonamide anticancer inhibitors of carbonic anhydrase IX exhibit lower toxic effects on zebrafish embryonic development than ethoxzolamide.</a> Drug and Chemical Toxicology. 40 (3), 309-319 (2017).</li><li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Granato, M., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+genes+controlling+development.">Fishing for genes controlling development.</a> Current Opinion in Genetics &amp; Development. 6 (4), 461-468 (1996).</li><li>Bambino, K., Chu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+in+Toxicology+and+Environmental+Health.">Zebrafish in Toxicology and Environmental Health.</a> Current Topics in Developmental Biology. 124, 331-367 (2017).</li><li>Goldsmith, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+pharmacological+tool:+the+how,+why+and+when.">Zebrafish as a pharmacological tool: the how, why and when.</a> Current Opinion in Pharmacology. 4 (5), 504-512 (2004).</li></ol>

In vitro toxicity test using cultured cells can detect survival and morphological studies of the cells providing limited information about the toxicity induced by the test compound. The advantage of toxicity screening of chemical compounds using zebrafish embryos is rapid detection of chemically induced phenotypic changes in a whole animal during embryonic development in a relevant model organism. Approximately 70% of protein-coding human genes have orthologs counterparts in the zebrafish genome<sup class="xref"...

Drug development is an expensive process. Before a single chemical compound is approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) several thousand compounds are screened at a cost of over one billion dollars1. During the preclinical development, the largest part of this cost is required for the animal testing2. To limit the costs, researchers in the field of drug development need alternative models for the safety screening of chemical compounds3. Therefore, in the early phase of the drug development, it would be very beneficial to use a method that can rapidly evaluate the safety and toxicity of the compounds in a suitable model. There are several protocols that have been used for the toxicity screening of chemical compounds involving animal and cell culture models but there is not a single protocol that is validated and is in common use4,5. Existing protocols using zebrafish vary in length and have been used by individual researchers who evaluated the toxicity as per their convenience requirement6,7,8,9,10,11,12.
In the recent past, the zebrafish has emerged as a convenient model for the evaluation of the toxicity of chemical compounds during embryonic development6,7. The zebrafish has many in-built advantages for the evaluation of chemical compounds13. Even large-scale experiments are amenable, as a zebrafish female can lay batches of 200-300 eggs, which develop rapidly ex&#160;vivo, do not need external feeding for up to a week and are transparent. The compounds can be added directly into the water, where they can (depending on the nature of the compound) diffuse through the chorion, and after hatching, through the skin, gills and mouth of larvae. The experiments do not require copious amounts of chemical compounds14 due to the small size of the embryo. Developing zebrafish embryos express most of the proteins required to achieve the normal developmental outcome. Therefore, a zebrafish embryo is a sensitive model to assess whether a potential drug can disturb the function of a protein or signaling molecule that is developmentally significant. The organs of the zebrafish become functional between 2-5 dpf15, and compounds that are toxic during this sensitive period of embryonic development induce phenotypic defects in zebrafish larvae. These phenotypic changes can be readily detected using a simple microscope without invasive techniques11. Zebrafish embryos are widely used in toxicological research due to their much greater biological complexity compared to in vitro drug screening using cell culture models16,17. &#160;As a vertebrate, the genetic and physiologic makeup of zebrafish is comparable to humans and hence toxicities of chemical compounds are similar between zebrafish and humans8,18,19,20,21,22. Zebrafish is, thus, a valuable tool in the early phase of drug discovery for the evaluation of toxicity and safety of the chemical compounds.
In the present article, we provide a detailed description of the method used for evaluating the safety and toxicity of carbonic anhydrase (CA) inhibitor compounds using 1-5-day post fertilization (dpf) zebrafish embryos by a single researcher. The protocol involves exposing zebrafish embryos to different concentrations of chemical inhibitor compounds and studying the mortality and phenotypic changes during the embryonic development. At the end of the exposure to the chemical compounds, the LC50 dose of the chemical is determined. The method allows an individual to carry out efficient screening of 1-5 test compounds and takes about 8-10 h depending on the experience of the person with the method (Figure 1). Each of the steps required to assess the toxicity of the compounds is outlined in Figure 2. The evaluation of toxicity of CA inhibitors requires 8 days, and includes setting up of mating pairs (day 1); collection of embryos from breeding tanks, cleaning and transferring them to 28.5 &#176;C incubator (day 2); distribution of the embryos into the wells of a 24-well plate and addition of diluted CA inhibitor compounds (day 3); phenotypic analysis and imaging of larvae (day 4-8), and determination of LC50 dose (day8). &#160;This method is rapid and efficient, requires a small amount of the chemical compound and only basic facilities of the laboratory.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">24-well plates</td>
			<td style="width:185px;">Nunc</td>
			<td style="width:260px;">Thermo Scientific</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Balance (Weighing scale)</td>
			<td style="width:185px;">KERN</td>
			<td style="width:260px;">PLJ3000-2CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Balance (Weighing scale)</td>
			<td style="width:185px;">Mettler Toledo</td>
			<td style="width:260px;">AB104-S/PH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">CaCl2</td>
			<td style="width:185px;">JT.Baker</td>
			<td style="width:260px;">RS421910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Disecting Probe</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td>17-467-604&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">DMSO</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">D4540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Falcon tubes 15 mL</td>
			<td style="width:185px;">Greiner bio-one</td>
			<td style="width:260px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">High molecular weight methylcellulose</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">M0262&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Incubator for zebrafish larvae</td>
			<td style="width:185px;">Termaks</td>
			<td style="width:260px;">B8000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">KCL</td>
			<td style="width:185px;">Merck</td>
			<td style="width:260px;">1.04936.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Methyl Blue</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">28983-56-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">MgSO4</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Microcentrifuge tubes</td>
			<td style="width:185px;">Starlab</td>
			<td style="width:260px;">S1615-5500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">NaCl</td>
			<td style="width:185px;">VWR Chemicals</td>
			<td style="width:260px;">27810.295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Paraffin Histoplast IM</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:260px;">8331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Pasteur pipette&#160;</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:260px;">86.1171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Petri dish</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td>101R20&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Petri plates</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:260px;">82.1473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Pipette (1 mL and 200 &#956;L)</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:260px;">4641230N, 4641210N&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Plates 24-Well</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:260px;">142485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Steriomicroscope/Camera</td>
			<td style="width:185px;">Zeiss</td>
			<td style="width:260px;">Stemi 2000-C/Axiocam 105 color</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Vials (1.5 mL)</td>
			<td style="width:185px;">Fisherbrand</td>
			<td style="width:260px;">11569914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Zebrafish AB strains</td>
			<td style="width:185px;">ZIRC&#160;</td>
			<td style="width:260px;">&#160; ZL1&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid evaluation of toxicity of chemical compounds using zebrafish embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

The critical part of the evaluation of toxicity is testing different concentrations of one or multiple chemical compounds in a single experiment. In the beginning, select the compounds for evaluation of toxicity, the number of concentrations to test for each compound, and accordingly, make a chart (Figure 3). We used a unique color for each compound to organize the samples (Figure 3). The use of solvent resistant marker and label...

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Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

The toxicity testing procedures will be demonstrated by Dr.Ashok Aspatwar, a senior researcher from my laboratory. Our protocol identifies off-target effects and detects subtle toxic effects of chemicals in the early phase of discovery that may be missed in cell culture or other animal models. The main advantages of our technique are:it is rapid and efficient, requires very small amount of chemical, and the basic facilities in the laboratory.Toxicity screening will help us in deciding the concentration for studying its efficiency against mycobacterium that causes tuberculosis and for further preclinical calculation. This method uses insights into off-target effects of chemicals, and hence this method can be used in any area of research where the toxicity of chemical is of primary importance. This method is very simple and can be carried out by anybody without prior experience.Individuals with no experience should plan the experiment carefully and use just one compound. This method provides toxic effects of the chemicals in the form of phenotypic changes in the developing zebrafish embryos and hence safety of the compounds tested. To begin, place two to five adult male zebrafish and three to five adult female zebrafish into mating tanks overnight.In the morning, turn on the light as breeding is induced by the automatic dark and subsequent light cycle. To avoid handling stress to the animals, allow the animals to rest for two weeks before using the same individuals for breeding. The next day before noon, collect the embryos using a fine mesh strainer, and transfer them onto a petri dish containing E3 embryo medium.Place the petri dish under a stereo microscope to examine each batch of embryos. Identify the opaque appearance and remove them as unfertilized or dead embryos. Keep the embryos at 28.5 degrees Celsius in an incubator overnight.The next morning, examine the embryos under a stereo microscope and remove any unhealthy or dead embryos. Carefully use a pasteur pipette to transfer one embryo into each well of a 24 well plate. Make sure each well contains enough E3 medium to cover the embryos.Take out the vials containing inhibitor compounds stored at 4 degrees Celsius in a refrigerator. Use an analytical balance to weigh appropriate amount of the compound, and prepare 250 microliters of 100 millimolar stock solution for each compound in E3 medium or another appropriate solvent. Then, use E3 medium to make serial dilutions of the stock solutions based on toxicity levels in 15 milliliter centrifuge tubes.From the wells that each contains one DPF embryo, use a pasteur pipette and 1 milliliter pipette to remove the E3 water one row at a time. Immediately distribute 1 milliliter of each diluent of the stock solutions into the wells of the 24 well plate starting from lower and moving to higher concentration. For the control groups, add E3 water or another relevant solvent.Label 24 well plates with the name and concentration of the compound and keep the plates at 28.5 degrees Celsius in an incubator. Take care that the dilutants of the chemicals in the wells is not evaporated in the incubator by sealing the sides of 24 well plate. Twenty four hours after exposure of the chemical compounds, use a pasteur pipette to transfer the larvae exposed to each concentration of the compound in a small petri dish containing 3%high molecular weight methyl cellulose.With a metal probe, lay it sideways. Place the petri dish under a stereo microscope attached to a camera. Take the images, and save the images in a separate folder each day until the end of the experiment.Enter all the observations in a table each day either in an online table or on a printed sheet. For neurotoxic compound exposure, the four to five DPF larvae may show abnormal swim pattern. In that case, make a record of such changes by capturing a short 30 seconds to one minute video of the larvae.After five days of exposure to the chemical compounds, note the concentration at which half of the embryos die as the half maximal lethal concentration 50 of each chemical. Construct a curve for mortality of embryos for all the concentrations using a suitable program. In this protocol, the critical part of the evaluation of toxicity is testing different concentrations of one or multiple chemical compounds in a single experiment.For the compounds that induce any phenotypic defects in the larvae, the defects were recorded every 24 hours over the period of one to five days post exposure to the chemical. The embryos treated with beta CA inhibitors at the concentrations of 250 micromolar and 125 micromolar exhibit various phenotypic defects compared with the control. For example, unhatched embryos even at Day 3, curved body structure, unutilized yolk sac and pericardial edema, and absence of otolith sacs in the larvae at five days after treatment.In another study, the embryos treated with CA inhibitors showed the absence of otolith sacs and swim bladder. The experiments identified a representative compound carbonic anhydrase 9, which induced minimal or no phenotypic changes during the embryonic development at 500 micromolar. The compound showed a high LC50 concentration.However, in a comparison with a control, the same compound at 300 micromolar was found to be neurotoxic and induced ataxia in the larvae after five days of exposure. Good quality of embryos are important for toxicity screening of chemicals. For obtaining good quality embryos, use young pair of adult zebrafish for breeding.The compounds that emerge as safe can be further characterized by performing relevant in vitro and in vivo experiments. In the field of antituberculosis drug development, this technique helped to set up further experiments for in vivo inhibition of mycobacterium marinum in zebrafish embryos. The protocol involves use of chemicals that may be toxic to humans.Persons involved in the experiments should take proper care when handling the chemicals.

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Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic properties allow them to dissolve hydrophobic compounds in the aqueous environment of the human body. However, self-assembling peptide solutions have poor blood compatibility (e.g., low osmolarity), hindering their clinical application through intravenous administrations. We have recently developed a generalized platform for hydrophobic drug delivery, which combines SAPs with amino acid solutions (SAP-AA) to enhance drug solubility and increase formulation osmolarity to reach the requirements for clinical uses. This formulation strategy was thoroughly tested in the context of three structurally different hydrophobic compounds &#8211; PP2, rottlerin, and curcumin &#8211; in order to demonstrate its versatility. Furthermore, we examined effects of changing formulation components by analyzing 6 different SAPs, 20 naturally existing amino acids at low and high concentrations, and two different co-solvents dimethyl sulfoxide (DMSO) and ethanol. Our strategy proved to be effective in optimizing components for a given hydrophobic drug, and therapeutic function of the formulated inhibitor, PP2, was observed both in vitro and in vivo. This manuscript outlines our generalized formulation method using SAP-AA combinations for hydrophobic compounds, and analysis of solubility as a first step towards potential use of these formulations in more functional studies. We include representative solubility results for formulation of the hydrophobic compound, curcumin, and discuss how our methodology serves as a platform for future biological studies and disease models.

This protocol describes a clinically-applicable means of dissolving hydrophobic compounds in an aqueous environment using combinations of self-assembling peptide and amino acid solutions. Our method resolves a major limitation of hydrophobic therapeutics, which lack safe, efficient means of solubility and delivery methods into clinical settings.

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...