One critical step in the development of a potential drug is the determination of its disposition in vivo prior to administration to humans. This is usually done soon after positive in vivo efficacy data has been obtained in rodents and is often required as part of IND (investigative new drug) filing. Preliminary pharmacokinetic (PK) studies used to determine project feasibility are relatively inexpensive and can be readily accomplished with a small amount of radioactively labeled compound synthesized by experienced companies such as TriLink BioTechnologies.
Radioactively labeled oligonucleotides are an ideal choice for PK studies because radioisotopes have virtually no affect on the functionality of the biomolecule. Substitution of a radioactive label in place of its "cold" analog is the least intrusive means of adding a tracer to the molecule when key considerations are addressed during synthesis. The primary consideration for the use of a radioactively labeled oligonucleotide in vivo is to make sure the label is non-exchangeable. This enables tracking of the drug and not the label itself. Further, the label must also be in a location useful for determining the metabolism of the drug. Finally, careful consideration of the quantity of material needed is also important.
Determining the best radioactive label for PK studies
The choice of label from TriLink is restricted to two isotopes: sulfur (35S) and tritium (3H). The best isotope for each PK study is determined by the specific needs of the experiment(s) being conducted. 35S has a higher energy and is therefore easier to detect at low levels, but it has a relatively short half-life of 88 days. Tritium, while being very long lived (12.4 years) is a relatively weak isotope, limiting levels of detection and analytical options. In addition to the half-life of the isotope and analysis requirements of the PK study, it is also important to consider the type of oligonucleotide that will be used. Isotopic sulfur obviously lends itself perfectly to use with phosphorothioate (PS) oligonucleotides, while tritium is better suited for non-PS oligonucleotides. Although a single radioactive phosphorothioate link can be added to any non-PS oligonucleotide, an overt change to the molecular composition will be made in doing so and this defeats one of the major advantage of using isotopes. Tritium, on the other hand, can be placed in any oligonucleotide, including phosphorothioates, with no change to molecular structure. TriLink offers a non-exchangeable tritium label on the 5′ position of thymidine. Other nucleosides can be procured, but they are considerably more expensive than thymidine.
TriLink has considerable experience preparing radioactively labeled oligonucleotides specifically for pre-clinical trials. We are happy to discuss your individual requirements and determine the isotope most appropriate for your research.
Determining the appropriate location of the radioactive label
The decision of where along the oligonucleotide to place the radio-label depends on the metabolism information required from the experiment, as well as the required yield. Yield, and hence price per mCi, is greatly affected by how far from the 5′ terminus the label is located. In general, the label should be found two to three bases from the 5' terminus for the best cost-to-effectiveness ratio. If there is protection against nuclease degradation at the 5′ terminus, then the label can be placed one or two bases from the terminus. The quantity of material needed to complete the PK studies should also be carefully considered. Simple PK studies in mice can be accomplished with as little as 1 µCi of tritium labeled oligonucleotide per animal. Total body tissue/organ distribution studies and blood clearance data can be rapidly determined with as little as 125 µCi of material, which is enough for a duplicate trial including 10 time points with 5 mice at each point. Larger studies will require up to 5 to 10 mCi of material, particularly if more precise information is required regarding the end result of the oligonucleotide in question.
For more information about the use of radioactively labeled oligonucleotides for PK studies, as well as literature about sample recovery and analysis, please see the bibliography below. If you have any questions, please do not hesitate to contact us.
References
Agrawal, S., Temsamani, J., & Tang, J.Y. Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice.(1991) Proc. National Acad. Sci. 88, 7595-7599
Agrawal, S., Zhang, X., Lu, Z., Zhao, H., Tamburin, J.M., Yan, J., Cai, H., Diasio, R.B., Habus, I., Jiang, Z., Iyer, R.P., Yu, D., & Zhang, R. Absorption, tissue distribution, and in vivo stability in rats of a hybrid antisense oligonucleotide following oral administration.(1995) Biochemical Pharmacology 50, (4):571-576
Beltinger, C., Saragovi, H.U., Smith, R.M., LeSauteur, L., Shah, N., DeDionisio, L., Christensen, L., Raible, A., Jarett, L., & Gewirtz, A.M. Binding, uptake, and intracellular trafficking of phosphorothioatemodified oligonucleotides.(1995) Journal of Clinical Investigation 95, (4):1814-1823
Cossum, P.A., Sasmor, H., Dellinger, D., Truong, L., Cummins, L., Owens, S.R., Markham, P.M., Shea, J.P., & Crooke, S. Disposition of the 14C-labeled phosphorothioate oligonucleotide ISIS 2105 after intravenous administration to rats.(1993) Journal of Pharm. and Exper. Therapeutics 267, (3):1181-1190
Crooke, R.M. Cellular uptake, distribution and metabolism of phosphorothioate, phosphodiester, and methylphosphonate oligonucleotides.(1997) in Antisense Research and Applications chapter 24, 427-449
De Serres, M., McNulty, M.J., Christensen, L., Zon, G., & Findlay, J.W.A. Development of a Novel Scintillation Proximity Competitive Hybridization Assay for the Determination of Phosphorothioate Antisense Oligonucleotide Plasma Concentrations in a Toxicokinetic Study.(1996) Analytical Biochemistry 233, 228-233
Iversen, P.L., Mata, J., Tracewell, W.G., & Zon, G. Pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in the adult male rat following single injections and continuous infusion.(1994) Antisense Research and Development 4, 43-52
Saijo, Y., Perlaky, L., Wang, H., & Busch, H. Pharmacokinetics, tissue distribution, and stability of antisense oligodeoxynucleotide phosphorothioate ISIS 3466 in mice.(1994) Oncology Research 6, (6):243-249
Sands, H., Gorey-Feret, L.J., Cocuzza, A.J., Hobbs, F.W., Chidester, D., & Trainor, G.L. Biodistribution and metabolism of internally 3Hlabeled oligonucleotides. Comparison of a phosphodiester and a phosphorothioate.( 1994) Molecular Pharmacology 45, 932-943
Sands, H., Gorey-Feret, L.J., Peng Ho, S., Cocuzza, A.J., Chidester, D., & Hobbs, F.W. Biodistribution and metabolism of internally 3H-labeled oligonucleotides. 3',5'-blocked oligonucleotides.(1995) Molecular Pharmacology 47, 636-646
Srinivasan, S.K., & Iversen, P.L. Review of in vivo pharmacokinetics and toxicology of phosphorothioate oligonucleotides.(1995) Journal of Clinical Laboratory Analysis 9, 129-137
Vaghefi, M.M., Fazio, R.C., Young, K.M., & Marvin, W.B. Synthesis of 3H-labeled nucleoside-methyl[CT3]phosphoramidite and incorporation into methylphosphonate oligonucleotides for biodistribution and biostability studies. (1995) Nucleic Acids Research 23, (17):3600-3602
Wu, D., Boado, R.J., & Pardridge, W.M. Pharmacokinetics and blood-brain barrier transport of [3H]-biotinylated phosphorothioate oligodeoxynucleotide conjugated to a vector-mediated drug delivery system.(1996) J. of Pharm. and Exp. Therapeutics 276, (1):206-211
Zhang, R., Lu, Z., Zhao, H., Zhang, X., Diasio, R.B., Habus, I., Jiang, Z., Iyer, R.P., Yu, D., & Agrawal, S. In vivo stability, disposition and metabolism of a "hybrid" oligonucleotide phosphorothioate in rats.(1995) Biochemical Pharmacology 50, (4):545-556
Zhang, R., Yan, J., Shahinian, H., Amin, G., Lu, Z., Liu, T., Saag, M.S., Jiang, Z., Temsamani, J., Martin, R.R., Schechter, P.J., Agrawal, S., & Diasio, R.B. Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM 91) in HIV-infected subjects.(1995) Clinical Pharmacology and Therapeutics 58, (1):44-53.
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