Abstract
Purpose. The therapeutic use of antisense oligonucleotides will likely involve their administration over protracted periods of time. The oral route of drug dosing offers many advantages over other possible routes when chronic drug administration is necessary. However, little is known about the potential for oligonucleotide uptake from the gastrointestinal tract. This issue is addressed in the current work.
Methods. We have developed a simple procedure for radiolabeling oligonucleotides by reductive alkylation with 14C-formaldehyde. We have utilized this approach, as well as 5′ addition of fluorophores, to prepare labeled methylphosphonate and phosphorothioate oligonucleotides for use in intestinal transport studies. An everted rat gut sac model was employed to compare the transport of oligonucleotides to that of model compounds whose permeation properties are better understood.
Results. We demonstrate that both methylphosphonate and phosphorothioate oligonucleotides are passively transported across the intestinal epithelium, probably by a paracellular route. The rates of transport for both types of oligonucleotides were similar, and were significantly greater than that of the very high MW polymer blue dextran, but were lower than the transport rate of valproic acid, a low MW compound known to have high oral availability.
Conclusions. A significant degree of permeation of oligonucleotides across the gastrointestinal epithelium does occur, but it is still unclear whether this is sufficient to permit effective oral administration of oligonucleotides as drugs.
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REFERENCES
Zamecnik, P. C. and Stephenson, M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. 75: 280–284 (1978).
Crooke S. T. Therapeutic applications of oligonucleotides. Annu. Rev. Pharmacol. Toxicol. 32: 329–376 (1992).
Milligan, J. F., Matteucci, M. D., and Martin, J. C. Current concepts in antisense drug design. J. Med. Chem. 36: 1923–1935 (1993).
Akhtar, S. and Juliano, R. L. Cellular uptake and intracellular fate of antisense oligonucleotides. Trends in Cell Biol. 2: 139–144 (1992).
Chem T. L., Miller, P. S., Ts'o, P. O. P., Colvin O. M. Disposition and Metabolism of Oligodeoxynucleotide methylphosphonate following a single iv injection in mice. Drug Metab. and Dispos. 18: 815–818 (1990).
Iverson, P. L., Mata, J., Tracewell, W. G., and Zon, G. Pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in adult male rat following single injections and continuous infusion. Antisense Research and Development 4: 43–52 (1994).
Cossum, P. A., Sasmor, H., Dellinger, D., Trunog, L., Cummins, L., Owens, S. R., Markham, P. M., Shea, J. P., and Crooke, S. Disposition of 14-C labeled phosphorothioate oligonucleotide ISIS 2105 after intravenous administration to rats. J. Pharm. Exp. Ther. 267: 1181–1190 (1993).
Wickstrom, E. Strategies for administering targeted therapeutic oligodeoxynucleotides. Trends in Biotechnology. 10: 281–287 (1992).
Agrawal, S., Temsamani, J., and Tang, J. Y. Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice. Proc. Natl. Acad. Sci. 88: 7595–7599 (1981).
Ma, T. Y., Dyer, D. L., and Said, H. M. Human intestinal cell line Caco-2: a useful model for studying cellular and molecular regulation of biotin uptake. Biochim. Biophys. Acta. 1189: 81–88 (1994).
Pappenheimer, J. R., Dahl, C. E., Karnovsky, M. L., and Maggio, J. E. Intestinal absorption and excretion of octapeptides composed of D amino acids. Proc. Natl. Acad. Sci. 91: 1942–1945 (1994).
Almoguera, C., Shibata, D., Forrester, K., Martin, J., Arnheim, and Perucho, M. Most human carcinomas of the exocrine pancreas contain mutant c-Ki-ras. Cell. 53: 549–554 (1988).
Hogrefe, R. I., Reynolds, M. A., Vaghefi, M. M., Young, K. M., Riley, T. A., Klem, R. E., and Arnold, L. J. J. 1993. An improved method for the synthesis and deprotection of methylphosphonate oligonucleotides. In, Synthetic Chemistry of Oligonucleotides and Their Analogs. Agrawal, S. ed. Humana Press. Totowa, NJ.
Pento, J. T., and Mousissian, G. K. Time-dependent deterioration of active transport in duodenal segments of rat intestine. J. Pharmacol. Methods. 20: 9–14 (1988).
Nahata, M. C., Breech, L., Ailaboui, A., and Murray, R. D. Absorption of valproic acid from the gastrointestinal tract of the piglet. Eur. J. Drug Metab. Pharmacokinet. 17: 129–134 (1992).
Mehvar, R., and Shepard, T. L. Molecular-weight-dependent pharmacokinetics of fluorescein-labeled dextrans in rats. J. Pharm. Sci. 81: 908–912 (1992).
Sands, H., Gorey-Feret, L. J., Cocuzza, A. J., Hobbs, F. W., Chidester, D., and Trainor, G. L. Biodistribution and metabolism of internally 3H-labeled oligonucleotides. 1. Comparison of a phosphodiester and a phosphorothioate. Mol. Pharm. 45: 932–943 (1994).
Capaccioli, S., Di Pasquali, J., Mini, E., Mazzei, T., and Quattrone, A. Cationic lipids improve antisense oligonucleotide uptake and prevent degradation in cultured cells and in human serum. Biochem. Biophys. Res. Comm. 197: 818–825 (1993).
Graham, M. J., Freier, S. M., Crooke, R. M., Maslova, R. N., and Lesnik, E. A. Tritium labeling of antisense oligonucleotides by exchange with tritiated water. Nucl. Acid Res. 21: 3737–3743 (1993).
Chang, Y.-T. and Loew, G. H. Reaction mechanisms of formaldehyde with endocylic imino groups of nucleic acid bases. J. Am. Chem. Soc. 116: 3548–3555 (1994).
Pappenheimer, J. R. Biological regulation of transepithelial impedance in the intestinal mucosa of rats and hamsters. J. Membrane Biol. 100: 137–148 (1987).
Yamamoto, A., Sakane, T., Shibukawa, M., Hashisa, J., and Sezaki, H. Absorption and metabolic characteristics of p-aminobenzoic acid and its isomer m-aminobenzoic acid, for the rat small intestine. J. Pharm. Sci. 80: 1067–1071 (1991).
Terasaki, T., Takakuwa, S., Moritani, S., and Tsuji, A. Transport of monocarboxylic acids at the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells. J. Pharmacol. Exp. Ther. 258: 932–937 (1991).
Wiseman, G. Sac of everted intestine technique for study of intestinal absorption in vitro. Methods in Med. Res. 9: 287–292 (1961).
Binks, S. P. and Dobrota, M. Kinetics and mechanism of uptake of platinum-based pharmaceuticals by the rat small intestine. Biochem. Pharmacol. 40: 1329–1336 (1990).
Wilson, T. and Wiseman, G. The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J. Physiol. 123: 116–125 (1954).
Levine, R. R., McNary, W. F., Kornguth, P. J., and LeBlanc, R. Histological re-evaluation of everted gut technique for studying intestinal absorption. Euro. J. Pharm. 9: 211–219 (1970).
Gardner, M. L. G. The absorptive viability of isolated intestine prepared from dead animals. Quarterly Journal of Experimental Physiology. 63: 93–95 (1978).
Ekstrom, G. M., Westrom, B. R., Telemo, E., and Karlsson, B. W. The uptake of fluorescein-conjugated dextran 70,000 by the small intestinal epithelium of the young rat and pig in relation to macromolecular transmission into the blood. J. Devel. Physiol. 10: 227–233 (1988).
Bizios, R., Blumenstock, F. A., Del Vecchio, P. J., and Malik, A. B. Permselectivity of cultured endothelial monolayers. J. Cell Biol. 106: 192a (1986).
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Hughes, J.A., Avrutskaya, A.V., Brouwer, K.L.R. et al. Radiolabeling of Methylphosphonate and Phosphorothioate Oligonucleotides and Evaluation of Their Transport in Everted Rat Jejunum Sacs. Pharm Res 12, 817–824 (1995). https://doi.org/10.1023/A:1016296617434
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DOI: https://doi.org/10.1023/A:1016296617434