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Regeneration of the antivirel drug (E) -5-(2-bromovinyl)-2′ -deoxyuridine in vivo

Regeneration of the antivirel drug (E) -5-(2-bromovinyl)-2′ -deoxyuridine in vivo Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Volume 12 Number 4 1984 Nucleic Acids Research Regeneration of the anthiral drug (£>5-<2-bromoviny1)-2'-deoxyurtdine in vivo Claude Desgranges* + , Gabriel Razaka*, Francoise Drouillet*, Henri Bricaud*, Piet Herdewijn"1" and Erik De aerc q + •Unite 8 de Cardiologie de 1'INSERM, Avenue du Haut-Levgque, 33600 Pessac, France, and +Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium Received 5 January 1984; Accepted 23 January 1984 ABSTRACT The highly potent and selective antiherpes drug BVdUrd ((J0-5-(2-bromo- vinyl)-2'-deoxyuridine] is cleared within 2-3 hours from the bloodstream upon intraperitoneal administration to rats. It is degraded to BVUra ((E)~ 5-(2-bromovinyl)uracil) and this inactive metabolite is cleared very slowly from the bloodstream so that 2A hours after the administration of BVdUrd, BVUra is still detectable in the plasma. This contrasts with several other 5-substituted uracils, i.e. 5-fluorouracil, 5-iodouracil, 5-trifluorothymine and thymine itself, which are, like their 2'-deoxyuridine counterparts FdUrd, IdUrd, F3dThd and dThd, cleared from the plasma within 2-3 hours. The injec- tion of dThd or any of the other 5-substituted 2'-deoxyuridines at 3 hours after the injection of BVdUrd, that is at a time when BVdUrd has disappeared completely from the circulation, results in the re-apparition of BVdUrd in the plasma. Apparently, BVdUrd is regenerated from BVUra following the reac- tion catalyzed by pyrimidine nucleoside phosphorylases : BVUra + dThd •+ BVdUrd + Thy. BVdUrd can even be generated de novo if dThd (or FdUrd, IdUrd or F3dThd) are administered 3 hours after a preceding injection of BVUra. These findings represent a unique example of the (re)generation of an active drug from its inactive metabolite in vivo. INTRODUCTION BVdUrd ((Ji)-5-(2-broniovinyl)-2'-deoxyuridine) is a highly potent and selective antiherpes agent (1), which is particularly effective against her- pes simplex virus type 1 (HSV-1) (2) and varicella zoster virus (VZV) (3). BVdUrd holds great promise for the treatment of HSV-1 and VZV infections in humans (4-7). The selectivity of BVdUrd as an antiviral drug primarily de- pends on a specific phosphorylation by the virus-induced thymidine (dThd) kinase, which restricts the further action of the compound to the virus-in- fected cell (8,9). Upon conversion to the 5'-triphoshate, BVdUrd may either interfere with viral DNA polymerase (10) or be incorporated into viral DNA (11,12), thereby disturbing the normal functioning of this DNA. Because of its highly potent and selective action against HSV-1 and VZV, BVdUrd is being actively pursued as a chemotherapeutic means to treat HSV-1 and VZV infections in humans. However, like other dUrd analogues, BVdUrd acts © IR L Press Limited, Oxford, England. 2081 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research as substrate for dThd phosphorylase (13) and uridine (Urd) phosphorylase (our unpublished observations) which cleave the compound at its N-glycosidic lin- kage, thereby releasing the inactive metabolite of BVdUrd, namely BVUra ((E)-5-(2-bromovinyl)uracil)• This cleavage would also occur rn vivo, since BVdUrd is rapidly cleared from the bloodstream (14) where it is replaced by BVUra, as demonstrated particularly in humans by R.J. Whitley e_t jil_. (perso- nal communication). The rapid degradation of BVdUrd may obviously affect the therapeutic usefulness of the drug, and, therefore, attempts should be under- taken to prevent this degradation. The molecule could be made more resistant to phosphorolytic cleavage by chemical modification of its structure or addi- tion of pyrimidine nucleoside phosphorylase inhibitors. However, previous attempts to decrease degradation of pyrimidine nucleosides by these proce- dures have not been very successful (15-17). In this paper we introduce a totally new approach to augment the effi- cacy of BVdUrd in vivo. This approach is based upon the regeneration of BVdUrd from its degradation product, BVUra. In contrast with BVdUrd, BVUra has a long half-time life in the bloodstream. Thus, we considered the possi- bility of restoring BVdUrd from BVUra via a pentosyl transfer reaction cata- lysed by dThd or Urd phosphorylases (18-20). As shown here, BVdUrd can indeed be generated in vivo from BVUra, i.e. upon intraperitoneal injection of dThd and other 5-substituted dUrd derivatives in rats. MATERIALS AND METHODS Products dThd, 5-iodo-dUrd (IdUrd), 5-fluoro-dUrd (FdUrd) and 5-trifluoro-dThd (F^dlhd) were obtained from Sigma Chemical Co., St. Louis, Missouri. BVdUrd was synthesized according to Jones et al. (21). BVUra was obtained by oxida- tive degradation of 5'-trityl-BVdUrd with dipyridine chromium (VI) oxide. 6-aminothymine (6-amino-Thy) was prepared by alkaline cyclisation of a-me- thyl-cyanoacetyl-urea according to the procedure of Bergmann and Johnson (22). Trioctylamine and Freon were purchased from E. Merck, Darmstadt, Federal Re- public of Germany. Pharmacokinetics Nucleosides and bases were dissolved or suspended in sterile physiologi- cal saline at a concentration of 90 pmoles/ml. One ml of these solutions was administered intraperitoneally (200 umoles/kg) to adult male Wistar rats weighing approximately A50 g. The schedule of administration is described in the legends to the Figures. At various time intervals, one-ml blood samples 2082 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research F,dThd Figure 1. Plasma levels of BVdUrd, dThd, IdUrd, FdUrd and F^dThd and their bases following i.p. administration of the nucleosides to rats. 90 pinoles of the nucleoside analogs were given intraperitoneally to male Wistar rats weighing approximately 450 g. Blood was taken by venipuncture and plasma was collected after centrifugation. Plasma proteins were eliminated by PCA treat- ment. The pyrimidine nucleosides were separated from their corresponding ba- ses and quantitated by HPLC. The upper diagrams represent the nucleoside con- centrations and lower diagrams the corresponding base concentrations after administration of BVdUrd, dThd, IdUrd, FdUrd and F dThd. were obtained from the jugular vein and collected in precooled tubes contai- ning sodium citrate as the anticoagulant. The tubes were centrifuged 20 min at 1200 g at 4°C. The supernatant plasma was treated with two volumes of cold 6 X trichloroacetic acid (TCA) and the precipitated proteins were remo- ved by centrifugation. The TCA was removed by treatment of the samples with an equal volume of 0.5 M trioctylamine in Freon according to Khym (23). After centrifugation, 100 yl of the upper aqueous phase, which contained the nu- cleosides and bases, were analysed by high performance liquid chromatography (HPLC) on a reverse phase Radial Pak C18 column (Waters) in a Waters HPLC system equiped with a M-450 detector and a peak integrator. The mobile phase was a linear gradient of 10 mM potassium phosphate (pH 5.5) and methanol- potassium phosphate buffer (80:20), as described by Hartwick and Brown (24), except that the slope of the gradient was chosen according to the nature of the nucleosides and bases present in the sample. The peaks were identified on the basis of their retention times and quantitated by comparison with known standards. In vitro transfer reactions Direct pentosyl transfer was assayed in a reaction mixture that contai- 2083 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research BVUra • dThd =s= BVdUrd* Thy BVUra • FdUrd =s= BVdUrd + FUre t houri 0 BVUra • F dThd =s= BVdUrd + F^Thy BVUra + IdUrd =s= BVdUrd + lUra -o - • 20 20 0 1 2 3 4 0 1 2 3 1. T i m t f hours ) Figure 2. In vitro BVdUrd formation by direct or indirect pentosyl transfer from either dThd, FdUrd, IdUrd or F^dThd onto BVUra. BVdUrd synthesis cata- lyzed by human dThd phosphorylase was followed by direct pentosyl transfer (o o) or by indirect pentosyl transfer in the presence of 0.1 mM (• • ) , 1 mM (p D) and 10 mM (• •) potassium phosphate. ned 10 mM Tris-HCl pH 7.4, 0.1 mM BVUra, 0.1 mM dThd (or IdUrd, FdUrd, F,dThd) and human platelet dThd phosphorylase (20). Incubation was at 37°C for different times. Reaction products were separated and quantitated by HPLC as described above. Indirect pentosyl transfer (in the presence of phos- phate) was assayed in the same conditions as the direct transfer except that the reaction mixture contained 10, 1 or 0.1 mM potassium phosphate in addi- tion to the other ingredients. RESULTS When BVdUrd was administered intraperitoneally (i.p.) as a single dose of 30 mg (90 ymoles) per rat, it reached its maximum plasma concentration (approximately 70 pM) at 20 min after the injection. At 3 hr after the injec- tion it was completely cleared from the plasma (Fig. 1). In the mean time, the plasma concentration of BVUra raised to 50-60 pM and this level was main- tained for up to 7 hr. Even at 24 hr after BVdUrd administration, there was still a plasma BVUra level of 10-20 uM. The other pyrimidine deoxyribonucleo- sides, dThd, IdUrd, FdUrd and F,dThd were cleared from the plasma at a simi- 2084 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research IdUrd 100-1 FdUrd 100-j FdThd BVUra 100- BVUra 100- BVUra 50- 50- ft BVdUrd BVdUrd BVdUrd BVdUrd 40- to- 20- 20- 20- / V • 0 0- 0- 4 0 2 4 Tim e ( hours } Figure 3. Regeneration of BVdUrd after its total clearance from the blood. All rats received an i.p. injection of 90 pmoles BVdUrd. Three hours later, when BVdUrd had disappeared from the plasma, the rats received an i.p. in- jection of either dThd, IdUrd, FdUrd or F3dThd. The plasma concentrations of dThd, IdUrd, FdUrd and F3dThd (upper diagrams), the corresponding concentra- tions of BVUra (middle diagrams) and corresponding concentrations of regene- rated BVdUrd (lower diagrams) were followed as a function of time, as descri- bed in the legend to Fig. 1. Zero time corresponds to the injection of dThd, IdUrd, FdUrd or F3dThd, 3 hours after BVdUrd administration. Dotted lines represent the concentrations of dThd, IdUrd, FdUrd and F3dThd administered alone (upper diagrams) and the concentrations of BVUra following BVdUrd ad- ministration without the other nucleosides (middle diagrams). lar rate as BVdUrd (Fig. 1). These compounds also gave rise to the free pyri- midine bases, Thy, IUra, FUra and F.Thy, but, in contrast with BVUra, these pyrimidines disappeared from the plasma within 2 to 4 hr after their appea- rance (Fig. 1). The finding that BVUra, unlike the other pyrimidines, persisted for a long time in the bloodstream, prompted us to attempt to reverse the phospho- rolytic cleavage and to regenerate BVdUrd. Indeed, pyrimidine deoxyribonu- cleosides can be formed from their corresponding bases by pyrimidine nucleo- side phoaphorylases, i.e. dThd and Urd phosphorylases) (18-20). Such pentosyl transfer reactions occur readily jji vitro between BVUra and either dThd, IdUrd, FdUrd or F,dThd in the presence of purified dThd phosphorylaBe as de- 2085 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research 100- IdUrd ioo- IdUrd (in presence of 6-ammo-Thy) 50- 50- BVdUrd BVdUrd 20- 20- 0 0 0 2 U 0 2 <• Tim e ( hours) Figure 4. Inhibition of BVdUrd regeneration by 6-amino-Thy. Three hours af- ter the i.p. injection of BVdUrd (90 umoles), one rat received an i.p. injec- tion of 90 pinoles of IdUrd and another rat a mixture of 90 pmoles IdUrd and of 100 umoles 6-amino-Thy, a potent inhibitor of pyrimidine nucleoside phos- phorylases). The reappearance of BVdUrd was followed as explained in the le- gend to Fig. 3. During the observation period, 6-amino-Thy concentrations decreased from 50 pM at time 0 to 25 uM. monstrated in Fig. 2. In attempts to reverse the degradation of BVdUrd jji vivo, dThd, IdUrd, FdUrd or F,dThd were administered i.p. at 90 pinoles per rat 3 hr after the i.p. injection of 90 umoles BVdUrd, that is at a time when BVdUrd had disap- peared completely from the circulation. BVdUrd promptly reappeared in the plasma following the administration of dThd, IdUrd, FdUrd or F,dThd (Fig. 3) , thereby attaining a peak level of 40 pM (following administration of dThd). Concomitantly with the reappearance of BVdUrd, BVUra showed a decrease in plasma concentration (Fig. 3). These findings suggested that BVdUrd was re- generated from BVUra via transfer of the deoxyribosyl moiety from dThd, IdUrd, FdUrd or F.dThd. Further support for the regeneration of BVdUrd via the pentosyl transfer by pyrimidine nucleoside phosphorylases stemmed from the use of 6-amino-Thy, a potent inhibitor of these enzymes (13,17,25-27). If co-administered with IdUrd, 3 hr after the injection of BVdUrd, 6-amino-Thy increased the plasma level of IdUrd, while suppressing the reapparation of BVdUrd (Fig. 4) . This could be expected if 6-amino-Thy blocked the pentosyl transfer from IdUrd to BVUra by pyrimidine nucleoside phosphorylases. To unequivocally establish that BVdUrd could be generated d£ novo, BVUra 2086 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research 100-1 dTh d 100-1 IdUrd F dThd Figure 5. De novo generation of BVdUrd after administration of BVUra follo- wed by either dThd, IdUrd, FdUrd or F3dThd. All rats received an l.p. injec- tion of 90 ymoles BVUra. Three hours later (zero time on the diagrams), 90 ymoles of dThd, IdUrd, FdUrd or F3dThd were administered, and their plasma concentrations were followed as a function of time (upper diagrams). The concomitant BVUra concentrations (middle diagrams) and BVdUrd concentrations (lower diagrams) were also followed. Dotted lines represent the concentra- tions of dThd, IdUrd, FdUrd and F3dThd administered alone (upper diagrams) or BVUra administered alone (middle diagrams). itself was administered i.p. at 90 ymoles per rat, followed 3 hr later by the i.p. injection of either dThd, IdUrd, FdUrd or F,dThd (Fig. 5). The plasma BVUra levels achieved 3 hr after the administration of BVUra were comprised between 50 and 80 yM. Upon injection of dThd, IdUrd, FdUrd, or F.dThd, there was a prompt decrease in the plasma BVUra levels, concomitantly with the ap- parition of BVdUrd in the bloodstream (Fig. 5) . Under these experimental con- ditions, BVdUrd must have been made from its metabolite BVUra, thus generated de novo. DISCUSSION From our findings it is clear that the degradation of BVdUrd is fully reversible and that BVdUrd can be restored from its degradation product, BVUra, upon administration of other pyrimidine deoxyribosides. This proce- 2087 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research dure could be repeated several times after a single administration of BVdUrd (our unpublished results), since high levels of BVUra are maintained in the plasma for at least 24 hr. The reversible conversion of the free pyrimidine base to the deoxynucleoside may also occur with bases other than BVUra, but it is most elegantly demonstrated with BVUra because this pyrimidine analo- gue persists for a much longer time in the bloodstream than the other pyri- midines. From our findings it also appears that an inactive compound (BVUra) can be transformed i^a vivo to a potent chemotherapeutic agent (BVdUrd) . This transformation is based upon a pentosyl exchange reaction catalyzed by a py- rimidine nucleoside phosphorylase. The site of the regeneration of BVdUrd from BVUra remains to be elucidated. However, since the liver has been con- sidered as the primary site for the degradation of dThd and dThd analogues in vivo (28,29), it is likely that this regeneration takes place in the liver tissue. It also remains to be established whether the deoxyribosyl transfer is direct, and in this case specific for dThd phosphorylase, or indirect, because of the presence of phosphate in the cells where the exchange reaction occurs. Nevertheless, this study clearly shows that in vivo pyrimidine nu- cleoside phosphorylases act in two directions which may either decrease the therapeutic efficacy of a nucleoside (due to phosphorolysis to the inactive base) or enhance it (due to the regeneration of the active nucleoside from the inactive base). For therapeutic purposes, the nucleoside used as deoxyribosyl donor could be selected according to the following criteria : (i) the nature of the pyrimidine C-5 substituent, which may determine the efficiency of the pento- syl exchange reaction and BVdUrd regeneration; (ii) the own properties, i.e. antiviral activity, of the selected pyrimidine nucleosides, which may be ad- ded to those achieved by the regenerated BVdUrd. Apart from the therapeutic implications for potentiating the clinical efficacy of BVdUrd in the treatment of herpesvirus infections, our observa- tions offer a unique example of the (re)generation of an active drug from its inactive metabolite in vivo. ACKNOWLEDGEMENTS This investigation was supported by grants from the Institut National de la Sant6 et de la Recherche M6dicale (INSERM), the University of Bordeaux II (contrat de recherche 204), CRESTA (Credit 80 P 6034), the Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek (Krediet no. 30048.75), and a 2088 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research fellowship award to CD . from the Flemish Commissariaat-Generaal voor de Internationale Culturele Samenwerking. We thank Mrs. I. Belloc for excellent technical assistance and Dr. R. Busson and Prof. H. Vanderhaeghe for the syn- thesis of BVdUrd. P. Herdewijn is a Senior Research Assistant of the Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek. REFERENCES 1. De Clercq, E., Descamps, J., De Somer, P., Barr, P.J., Jones, A.S. and Walker, R.T. (1979) Proc. Natl. Acad. Sci. USA 76, 2947-2951. 2. De Clercq, E., Descamps, J., Verhelst, G., Walker, R.T., Jones, A.S., Torrence, P.F. and Shugar, D. (1980) J. Infect. Dis. 141, 563-574. 3. Shigeta, S., Yokota, T., Iwabuchi, T., Baba, M., Konno, K., Ogata, M. and De Clercq, E. (1983) J. Infect. Dis. 147, 576-584. 4. De Clercq, E., De Greef, H., Wildiers, J., De Jonge, G., Drochmans, A., Descamps, J. and De Somer, P. (1980) Brit. Med. J. 281, 1178. 5. Maudgal, P.C., Dralands, L., Lamberts, L., De Clercq, E., Descamps, J. and Missotten, L. (1981) Bull. Soc. beige Ophtal. 193, 49-56. 6. Maudgal, P.C., Missotten, L., De Clercq, E., Descamps, J. and De Meuter, E. (1981) Albrecht von Graefes Arch. Klin. Ophthalmol. 216, 261-268. 7. Wildiers, J. and De Clercq, E. (1983) Eur. J. Cancer Clin. Oncol., in press. 8. Descamps, J. and De Clercq, E. (1981) J. Biol. Chem. 256, 5973-5976. 9. Cheng, Y.C., Dutschman, C , De Clercq, E., Jones, A.S., Rahim, S.G., Verhelst, G. and Walker, R.T. (1981) Mol. Pharmacol. 20, 230-233. 10. Allaudeen, H.S., Kozarich, J.W., Bertino, J.R. and De Clercq, E. (1981) Proc. Natl. Acad. Sci. USA 78, 2698-2702. 11. Allaudeen, H.S., Chen, M.S., Lee, J.J., De Clercq, E. and Prusoff, W.H. (1982) J. Biol. Chem. 257, 603-606. 12. Mancini, W.R., De Clercq, E. and Prusoff, W.H. (1983) J. Biol. Chem. 258, 792-795. 13. Desgranges, C , Razaka, G., Rabaud, M. , Bricaud, H. , Balzarini, J. and De Clercq, E. (1983) Biochem. Pharmacol. 32, 3583-3590. 14. De Clercq, E., Descamps, J., De Somer, P., Barr, P.J., Jones, A.S. and Walker, R.T. (1979) Antimicrob. Agents Chemother. 16, 234-236. 15. Ensminger, W.D. and Rosowsky, A. (1979) Biochem. Pharmacol. 28, 1541- 16. Rosowsky, A., Wright, J.E., Steele, G. and Kufe, D.W. (1981) Cancer Treat. Rep. 65, 93-99. 17. Langen, P., Etzold, G., Barwolff, D. and Preussel, B. (1967) Biochem. Pharmacol. 16, 1833-1837. 18. Gallo, R.C., Perry, S. and Breitman, T.R. (1967) J. Biol. Chem. 242, 5059-5068. 19. Kreniteky, T.A. (1968) J. Biol. Chem. 243, 2871-2875. 20. Desgranges, C , Razaka, C , Rabaud, M. and Bricaud, H. (1981) Biochim. Biophys. Acta 654, 211-218. 21. Jones, A.S., Verhelst, G. and Walker, R.T. (1979) Tetrahedron Lett. 45, 4415-4418. 22. Bergmann, W. and Johnson, T.B. (1933) J. Amer. Chem. Soc. 55, 1733-1735. 23. Khym, J.X. (1975) Clin. Chem. 21, 1245-1252. 24. Hartwick, R.A. and Brown, P.R. (1976) J. Chromat. 126, 679-691. 25. Baker, B.R. and Kelley, J.L. (1971) J. Med. Chem. 14, 812-816. 26. Woodman, P.W., Sarrif, A.M. and Heidelberger, C. (1980) Biochem. Pharma- col. 29, 1059-1063. 2089 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research 27. Desgranges, C , Razaka, G. Rabaud, M., Picard, P., Dupuch, F. and Bri- caud, H. (1982) Biochem. Pharmacol. 31, 2755-2759. 28. Ensminger, W.D. and Frei, E. Ill (1978) Clin. Pharmacol. Ther. 24, 610- 29. Ensminger, W.D., Rosowsky, A., Raso, V., Levin, D.C., Glode, H., Como, S., Steele, G. and Frei, E. Ill (1978) Cancer Res. 38, 3784-3794. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Regeneration of the antivirel drug (E) -5-(2-bromovinyl)-2′ -deoxyuridine in vivo

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Abstract

Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Volume 12 Number 4 1984 Nucleic Acids Research Regeneration of the anthiral drug (£>5-<2-bromoviny1)-2'-deoxyurtdine in vivo Claude Desgranges* + , Gabriel Razaka*, Francoise Drouillet*, Henri Bricaud*, Piet Herdewijn"1" and Erik De aerc q + •Unite 8 de Cardiologie de 1'INSERM, Avenue du Haut-Levgque, 33600 Pessac, France, and +Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium Received 5 January 1984; Accepted 23 January 1984 ABSTRACT The highly potent and selective antiherpes drug BVdUrd ((J0-5-(2-bromo- vinyl)-2'-deoxyuridine] is cleared within 2-3 hours from the bloodstream upon intraperitoneal administration to rats. It is degraded to BVUra ((E)~ 5-(2-bromovinyl)uracil) and this inactive metabolite is cleared very slowly from the bloodstream so that 2A hours after the administration of BVdUrd, BVUra is still detectable in the plasma. This contrasts with several other 5-substituted uracils, i.e. 5-fluorouracil, 5-iodouracil, 5-trifluorothymine and thymine itself, which are, like their 2'-deoxyuridine counterparts FdUrd, IdUrd, F3dThd and dThd, cleared from the plasma within 2-3 hours. The injec- tion of dThd or any of the other 5-substituted 2'-deoxyuridines at 3 hours after the injection of BVdUrd, that is at a time when BVdUrd has disappeared completely from the circulation, results in the re-apparition of BVdUrd in the plasma. Apparently, BVdUrd is regenerated from BVUra following the reac- tion catalyzed by pyrimidine nucleoside phosphorylases : BVUra + dThd •+ BVdUrd + Thy. BVdUrd can even be generated de novo if dThd (or FdUrd, IdUrd or F3dThd) are administered 3 hours after a preceding injection of BVUra. These findings represent a unique example of the (re)generation of an active drug from its inactive metabolite in vivo. INTRODUCTION BVdUrd ((Ji)-5-(2-broniovinyl)-2'-deoxyuridine) is a highly potent and selective antiherpes agent (1), which is particularly effective against her- pes simplex virus type 1 (HSV-1) (2) and varicella zoster virus (VZV) (3). BVdUrd holds great promise for the treatment of HSV-1 and VZV infections in humans (4-7). The selectivity of BVdUrd as an antiviral drug primarily de- pends on a specific phosphorylation by the virus-induced thymidine (dThd) kinase, which restricts the further action of the compound to the virus-in- fected cell (8,9). Upon conversion to the 5'-triphoshate, BVdUrd may either interfere with viral DNA polymerase (10) or be incorporated into viral DNA (11,12), thereby disturbing the normal functioning of this DNA. Because of its highly potent and selective action against HSV-1 and VZV, BVdUrd is being actively pursued as a chemotherapeutic means to treat HSV-1 and VZV infections in humans. However, like other dUrd analogues, BVdUrd acts © IR L Press Limited, Oxford, England. 2081 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research as substrate for dThd phosphorylase (13) and uridine (Urd) phosphorylase (our unpublished observations) which cleave the compound at its N-glycosidic lin- kage, thereby releasing the inactive metabolite of BVdUrd, namely BVUra ((E)-5-(2-bromovinyl)uracil)• This cleavage would also occur rn vivo, since BVdUrd is rapidly cleared from the bloodstream (14) where it is replaced by BVUra, as demonstrated particularly in humans by R.J. Whitley e_t jil_. (perso- nal communication). The rapid degradation of BVdUrd may obviously affect the therapeutic usefulness of the drug, and, therefore, attempts should be under- taken to prevent this degradation. The molecule could be made more resistant to phosphorolytic cleavage by chemical modification of its structure or addi- tion of pyrimidine nucleoside phosphorylase inhibitors. However, previous attempts to decrease degradation of pyrimidine nucleosides by these proce- dures have not been very successful (15-17). In this paper we introduce a totally new approach to augment the effi- cacy of BVdUrd in vivo. This approach is based upon the regeneration of BVdUrd from its degradation product, BVUra. In contrast with BVdUrd, BVUra has a long half-time life in the bloodstream. Thus, we considered the possi- bility of restoring BVdUrd from BVUra via a pentosyl transfer reaction cata- lysed by dThd or Urd phosphorylases (18-20). As shown here, BVdUrd can indeed be generated in vivo from BVUra, i.e. upon intraperitoneal injection of dThd and other 5-substituted dUrd derivatives in rats. MATERIALS AND METHODS Products dThd, 5-iodo-dUrd (IdUrd), 5-fluoro-dUrd (FdUrd) and 5-trifluoro-dThd (F^dlhd) were obtained from Sigma Chemical Co., St. Louis, Missouri. BVdUrd was synthesized according to Jones et al. (21). BVUra was obtained by oxida- tive degradation of 5'-trityl-BVdUrd with dipyridine chromium (VI) oxide. 6-aminothymine (6-amino-Thy) was prepared by alkaline cyclisation of a-me- thyl-cyanoacetyl-urea according to the procedure of Bergmann and Johnson (22). Trioctylamine and Freon were purchased from E. Merck, Darmstadt, Federal Re- public of Germany. Pharmacokinetics Nucleosides and bases were dissolved or suspended in sterile physiologi- cal saline at a concentration of 90 pmoles/ml. One ml of these solutions was administered intraperitoneally (200 umoles/kg) to adult male Wistar rats weighing approximately A50 g. The schedule of administration is described in the legends to the Figures. At various time intervals, one-ml blood samples 2082 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research F,dThd Figure 1. Plasma levels of BVdUrd, dThd, IdUrd, FdUrd and F^dThd and their bases following i.p. administration of the nucleosides to rats. 90 pinoles of the nucleoside analogs were given intraperitoneally to male Wistar rats weighing approximately 450 g. Blood was taken by venipuncture and plasma was collected after centrifugation. Plasma proteins were eliminated by PCA treat- ment. The pyrimidine nucleosides were separated from their corresponding ba- ses and quantitated by HPLC. The upper diagrams represent the nucleoside con- centrations and lower diagrams the corresponding base concentrations after administration of BVdUrd, dThd, IdUrd, FdUrd and F dThd. were obtained from the jugular vein and collected in precooled tubes contai- ning sodium citrate as the anticoagulant. The tubes were centrifuged 20 min at 1200 g at 4°C. The supernatant plasma was treated with two volumes of cold 6 X trichloroacetic acid (TCA) and the precipitated proteins were remo- ved by centrifugation. The TCA was removed by treatment of the samples with an equal volume of 0.5 M trioctylamine in Freon according to Khym (23). After centrifugation, 100 yl of the upper aqueous phase, which contained the nu- cleosides and bases, were analysed by high performance liquid chromatography (HPLC) on a reverse phase Radial Pak C18 column (Waters) in a Waters HPLC system equiped with a M-450 detector and a peak integrator. The mobile phase was a linear gradient of 10 mM potassium phosphate (pH 5.5) and methanol- potassium phosphate buffer (80:20), as described by Hartwick and Brown (24), except that the slope of the gradient was chosen according to the nature of the nucleosides and bases present in the sample. The peaks were identified on the basis of their retention times and quantitated by comparison with known standards. In vitro transfer reactions Direct pentosyl transfer was assayed in a reaction mixture that contai- 2083 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research BVUra • dThd =s= BVdUrd* Thy BVUra • FdUrd =s= BVdUrd + FUre t houri 0 BVUra • F dThd =s= BVdUrd + F^Thy BVUra + IdUrd =s= BVdUrd + lUra -o - • 20 20 0 1 2 3 4 0 1 2 3 1. T i m t f hours ) Figure 2. In vitro BVdUrd formation by direct or indirect pentosyl transfer from either dThd, FdUrd, IdUrd or F^dThd onto BVUra. BVdUrd synthesis cata- lyzed by human dThd phosphorylase was followed by direct pentosyl transfer (o o) or by indirect pentosyl transfer in the presence of 0.1 mM (• • ) , 1 mM (p D) and 10 mM (• •) potassium phosphate. ned 10 mM Tris-HCl pH 7.4, 0.1 mM BVUra, 0.1 mM dThd (or IdUrd, FdUrd, F,dThd) and human platelet dThd phosphorylase (20). Incubation was at 37°C for different times. Reaction products were separated and quantitated by HPLC as described above. Indirect pentosyl transfer (in the presence of phos- phate) was assayed in the same conditions as the direct transfer except that the reaction mixture contained 10, 1 or 0.1 mM potassium phosphate in addi- tion to the other ingredients. RESULTS When BVdUrd was administered intraperitoneally (i.p.) as a single dose of 30 mg (90 ymoles) per rat, it reached its maximum plasma concentration (approximately 70 pM) at 20 min after the injection. At 3 hr after the injec- tion it was completely cleared from the plasma (Fig. 1). In the mean time, the plasma concentration of BVUra raised to 50-60 pM and this level was main- tained for up to 7 hr. Even at 24 hr after BVdUrd administration, there was still a plasma BVUra level of 10-20 uM. The other pyrimidine deoxyribonucleo- sides, dThd, IdUrd, FdUrd and F,dThd were cleared from the plasma at a simi- 2084 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research IdUrd 100-1 FdUrd 100-j FdThd BVUra 100- BVUra 100- BVUra 50- 50- ft BVdUrd BVdUrd BVdUrd BVdUrd 40- to- 20- 20- 20- / V • 0 0- 0- 4 0 2 4 Tim e ( hours } Figure 3. Regeneration of BVdUrd after its total clearance from the blood. All rats received an i.p. injection of 90 pmoles BVdUrd. Three hours later, when BVdUrd had disappeared from the plasma, the rats received an i.p. in- jection of either dThd, IdUrd, FdUrd or F3dThd. The plasma concentrations of dThd, IdUrd, FdUrd and F3dThd (upper diagrams), the corresponding concentra- tions of BVUra (middle diagrams) and corresponding concentrations of regene- rated BVdUrd (lower diagrams) were followed as a function of time, as descri- bed in the legend to Fig. 1. Zero time corresponds to the injection of dThd, IdUrd, FdUrd or F3dThd, 3 hours after BVdUrd administration. Dotted lines represent the concentrations of dThd, IdUrd, FdUrd and F3dThd administered alone (upper diagrams) and the concentrations of BVUra following BVdUrd ad- ministration without the other nucleosides (middle diagrams). lar rate as BVdUrd (Fig. 1). These compounds also gave rise to the free pyri- midine bases, Thy, IUra, FUra and F.Thy, but, in contrast with BVUra, these pyrimidines disappeared from the plasma within 2 to 4 hr after their appea- rance (Fig. 1). The finding that BVUra, unlike the other pyrimidines, persisted for a long time in the bloodstream, prompted us to attempt to reverse the phospho- rolytic cleavage and to regenerate BVdUrd. Indeed, pyrimidine deoxyribonu- cleosides can be formed from their corresponding bases by pyrimidine nucleo- side phoaphorylases, i.e. dThd and Urd phosphorylases) (18-20). Such pentosyl transfer reactions occur readily jji vitro between BVUra and either dThd, IdUrd, FdUrd or F,dThd in the presence of purified dThd phosphorylaBe as de- 2085 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research 100- IdUrd ioo- IdUrd (in presence of 6-ammo-Thy) 50- 50- BVdUrd BVdUrd 20- 20- 0 0 0 2 U 0 2 <• Tim e ( hours) Figure 4. Inhibition of BVdUrd regeneration by 6-amino-Thy. Three hours af- ter the i.p. injection of BVdUrd (90 umoles), one rat received an i.p. injec- tion of 90 pinoles of IdUrd and another rat a mixture of 90 pmoles IdUrd and of 100 umoles 6-amino-Thy, a potent inhibitor of pyrimidine nucleoside phos- phorylases). The reappearance of BVdUrd was followed as explained in the le- gend to Fig. 3. During the observation period, 6-amino-Thy concentrations decreased from 50 pM at time 0 to 25 uM. monstrated in Fig. 2. In attempts to reverse the degradation of BVdUrd jji vivo, dThd, IdUrd, FdUrd or F,dThd were administered i.p. at 90 pinoles per rat 3 hr after the i.p. injection of 90 umoles BVdUrd, that is at a time when BVdUrd had disap- peared completely from the circulation. BVdUrd promptly reappeared in the plasma following the administration of dThd, IdUrd, FdUrd or F,dThd (Fig. 3) , thereby attaining a peak level of 40 pM (following administration of dThd). Concomitantly with the reappearance of BVdUrd, BVUra showed a decrease in plasma concentration (Fig. 3). These findings suggested that BVdUrd was re- generated from BVUra via transfer of the deoxyribosyl moiety from dThd, IdUrd, FdUrd or F.dThd. Further support for the regeneration of BVdUrd via the pentosyl transfer by pyrimidine nucleoside phosphorylases stemmed from the use of 6-amino-Thy, a potent inhibitor of these enzymes (13,17,25-27). If co-administered with IdUrd, 3 hr after the injection of BVdUrd, 6-amino-Thy increased the plasma level of IdUrd, while suppressing the reapparation of BVdUrd (Fig. 4) . This could be expected if 6-amino-Thy blocked the pentosyl transfer from IdUrd to BVUra by pyrimidine nucleoside phosphorylases. To unequivocally establish that BVdUrd could be generated d£ novo, BVUra 2086 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research 100-1 dTh d 100-1 IdUrd F dThd Figure 5. De novo generation of BVdUrd after administration of BVUra follo- wed by either dThd, IdUrd, FdUrd or F3dThd. All rats received an l.p. injec- tion of 90 ymoles BVUra. Three hours later (zero time on the diagrams), 90 ymoles of dThd, IdUrd, FdUrd or F3dThd were administered, and their plasma concentrations were followed as a function of time (upper diagrams). The concomitant BVUra concentrations (middle diagrams) and BVdUrd concentrations (lower diagrams) were also followed. Dotted lines represent the concentra- tions of dThd, IdUrd, FdUrd and F3dThd administered alone (upper diagrams) or BVUra administered alone (middle diagrams). itself was administered i.p. at 90 ymoles per rat, followed 3 hr later by the i.p. injection of either dThd, IdUrd, FdUrd or F,dThd (Fig. 5). The plasma BVUra levels achieved 3 hr after the administration of BVUra were comprised between 50 and 80 yM. Upon injection of dThd, IdUrd, FdUrd, or F.dThd, there was a prompt decrease in the plasma BVUra levels, concomitantly with the ap- parition of BVdUrd in the bloodstream (Fig. 5) . Under these experimental con- ditions, BVdUrd must have been made from its metabolite BVUra, thus generated de novo. DISCUSSION From our findings it is clear that the degradation of BVdUrd is fully reversible and that BVdUrd can be restored from its degradation product, BVUra, upon administration of other pyrimidine deoxyribosides. This proce- 2087 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research dure could be repeated several times after a single administration of BVdUrd (our unpublished results), since high levels of BVUra are maintained in the plasma for at least 24 hr. The reversible conversion of the free pyrimidine base to the deoxynucleoside may also occur with bases other than BVUra, but it is most elegantly demonstrated with BVUra because this pyrimidine analo- gue persists for a much longer time in the bloodstream than the other pyri- midines. From our findings it also appears that an inactive compound (BVUra) can be transformed i^a vivo to a potent chemotherapeutic agent (BVdUrd) . This transformation is based upon a pentosyl exchange reaction catalyzed by a py- rimidine nucleoside phosphorylase. The site of the regeneration of BVdUrd from BVUra remains to be elucidated. However, since the liver has been con- sidered as the primary site for the degradation of dThd and dThd analogues in vivo (28,29), it is likely that this regeneration takes place in the liver tissue. It also remains to be established whether the deoxyribosyl transfer is direct, and in this case specific for dThd phosphorylase, or indirect, because of the presence of phosphate in the cells where the exchange reaction occurs. Nevertheless, this study clearly shows that in vivo pyrimidine nu- cleoside phosphorylases act in two directions which may either decrease the therapeutic efficacy of a nucleoside (due to phosphorolysis to the inactive base) or enhance it (due to the regeneration of the active nucleoside from the inactive base). For therapeutic purposes, the nucleoside used as deoxyribosyl donor could be selected according to the following criteria : (i) the nature of the pyrimidine C-5 substituent, which may determine the efficiency of the pento- syl exchange reaction and BVdUrd regeneration; (ii) the own properties, i.e. antiviral activity, of the selected pyrimidine nucleosides, which may be ad- ded to those achieved by the regenerated BVdUrd. Apart from the therapeutic implications for potentiating the clinical efficacy of BVdUrd in the treatment of herpesvirus infections, our observa- tions offer a unique example of the (re)generation of an active drug from its inactive metabolite in vivo. ACKNOWLEDGEMENTS This investigation was supported by grants from the Institut National de la Sant6 et de la Recherche M6dicale (INSERM), the University of Bordeaux II (contrat de recherche 204), CRESTA (Credit 80 P 6034), the Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek (Krediet no. 30048.75), and a 2088 Downloaded from https://academic.oup.com/nar/article/12/4/2081/2378843 by DeepDyve user on 20 August 2020 Nucleic Acids Research fellowship award to CD . from the Flemish Commissariaat-Generaal voor de Internationale Culturele Samenwerking. We thank Mrs. I. Belloc for excellent technical assistance and Dr. R. Busson and Prof. H. Vanderhaeghe for the syn- thesis of BVdUrd. P. 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Nucleic Acids Research – Oxford University Press

Published: Feb 24, 1984

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