Amino Acids

, Volume 50, Issue 8, pp 1131–1143 | Cite as

Tailoring acyclovir prodrugs with enhanced antiviral activity: rational design, synthesis, human plasma stability and in vitro evaluation

  • Radoslav L. Chayrov
  • Evgenios K. Stylos
  • Maria V. Chatziathanasiadou
  • Kiril N. Chuchkov
  • Aleksandra I. Tencheva
  • Androniki D. Kostagianni
  • Tsenka S. Milkova
  • Assia L. Angelova
  • Angel S. Galabov
  • Stoyan A. Shishkov
  • Daniel G. Todorov
  • Andreas G. TzakosEmail author
  • Ivanka G. StankovaEmail author
Original Article


Bile acid prodrugs have served as a viable strategy for refining the pharmaceutical profile of parent drugs through utilizing bile acid transporters. A series of three ester prodrugs of the antiherpetic drug acyclovir (ACV) with the bile acids cholic, chenodeoxycholic and deoxycholic were synthesized and evaluated along with valacyclovir for their in vitro antiviral activity against herpes simplex viruses type 1 and type 2 (HSV-1, HSV-2). The in vitro antiviral activity of the three bile acid prodrugs was also evaluated against Epstein–Barr virus (EBV). Plasma stability assays, utilizing ultra-high performance liquid chromatography coupled with tandem mass spectrometry, in vitro cytotoxicity and inhibitory experiments were conducted in order to establish the biological profile of ACV prodrugs. The antiviral assays demonstrated that ACV-cholate had slightly better antiviral activity than ACV against HSV-1, while it presented an eight-fold higher activity with respect to ACV against HSV-2. ACV-chenodeoxycholate presented a six-fold higher antiviral activity against HSV-2 with respect to ACV. Concerning EBV, the highest antiviral effect was demonstrated by ACV-chenodeoxycholate. Human plasma stability assays revealed that ACV-deoxycholate was more stable than the other two prodrugs. These results suggest that decorating the core structure of ACV with bile acids could deliver prodrugs with amplified antiviral activity.


Acyclovir Prodrugs Plasma stability HSV-1 HSV-2 Epstein–Barr virus 





Herpes simplex virus type 1


Herpes simplex virus type 2


Epstein–Barr virus


Liquid chromatography–tandem mass spectrometry


Retention time


Multiple reaction monitoring


Maximum tolerable concentration


Minimum inhibitory concentration



We thank Dr. Regina Feederle from the German Cancer Research Center, Heidelberg for assistance with immunofluorescence staining, and Dr. Yavor Mitrev from Laboratory “Bulgarian NMR Centre” Institute of Organic Chemistry with Centre of Phytochemistry, Sofia for helpful discussions.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

All authors listed have contributed to conception, design, gathering, analysis, or interpretation of data, and have contributed to the writing and intellectual content of the article. All authors gave informed consent to the submission of this manuscript.


  1. Antman MD, Gudmundsson OS (2007) Case study: valacyclovir: a prodrug of acyclovir. In: Stella VJ, Borchardt RT, Hageman MJ, Oliyai R, Maag H, Tilley JW (eds) Prodrugs: challenges and rewards part 1. Springer, New York, pp 1369–1376. CrossRefGoogle Scholar
  2. Arkin LM, Castelo-Soccio L, Kovarik C (2009) Disseminated herpes simplex virus (HSV) hepatitis diagnosed by dermatology evaluation. Int J Dermatol 48(9):1020–1021. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Balakrishnan A, Polli JE (2006) Apical sodium dependent bile acid transporter (ASBT, SLC10A2): a potential prodrug target. Mol Pharm 3(3):223–230. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bando H, Yamashita F, Takakura Y, Hashida M (1994) Skin penetration enhancement of acyclovir by prodrug-enhancer combination. Biol Pharm Bull 17(8):1141–1143CrossRefPubMedGoogle Scholar
  5. Beauchamp LM, Krenitsky TA (1993) Acyclovir prodrugs: the road to valaciclovir. Drugs Future 18(7):619–628CrossRefGoogle Scholar
  6. Beauchamp LM, Orr GF, de Miranda P, Bumette T, Krenitsky TA (1992) Amino acid ester prodrugs of acyclovir. Antiviral Chem Chemother 3(3):157–164. CrossRefGoogle Scholar
  7. Berger JR, Houff S (2008) Neurological complications of herpes simplex virus type 2 infection. Arch Neurol 65(5):596–600. CrossRefPubMedGoogle Scholar
  8. Bockman DE, Ganapathy V, Oblak TG, Leibach FH (1997) Localization of peptide transporter in nuclei and lysosomes of the pancreas. Int J Pancreatol 22(3):221–225. PubMedCrossRefGoogle Scholar
  9. Bomgaars L, Thompson P, Berg S, Serabe B, Aleksic A, Blaney S (2008) Valacyclovir and acyclovir pharmacokinetics in immunocompromised children. Pediatr Blood Cancer 51(4):504–508. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Bras AP, Sitar DS, Aoki FY (2001) Comparative bioavailability of acyclovir from oral valacyclovir and acyclovir in patients treated for recurrent genital herpes simplex virus infection. Can J Clin Pharmacol 8(4):207–211PubMedGoogle Scholar
  11. Bruni G, Maietta M, Maggi L, Mustarelli P, Ferrara C, Berbenni V, Milanese C, Girella A, Marini A (2013) Preparation and physicochemical characterization of acyclovir cocrystals with improved dissolution properties. J Pharm Sci 102(11):4079–4086. CrossRefPubMedGoogle Scholar
  12. Burnette TC, de Miranda P (1994) Metabolic disposition of the acyclovir prodrug valaciclovir in the rat. Drug Metab Dispos 22(1):60–64PubMedGoogle Scholar
  13. Carey MC, Cahalane MJ (1988) In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritz DS (eds) In the liver: biology and pathobiology. Raven Press, New York, pp 573–616Google Scholar
  14. Chaudhary D, Ahmed S, Liu N, Marsano-Obando L (2017) Acute liver failure from herpes simplex virus in an immunocompetent patient due to direct inoculation of the peritoneum. ACG Case Rep J 4:e23. PubMedPubMedCentralCrossRefGoogle Scholar
  15. Chiang JY (2009) Bile acids: regulation of synthesis. J Lipid Res 50(10):1955–1966. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Colla L, De Clercq E, Busson R, Vanderhaeghe H (1983) Synthesis and antiviral activity of water-soluble esters of acyclovir [9-[(2-hydroxyethoxy)methyl]guanine). J Med Chem 26(4):602–604. CrossRefPubMedGoogle Scholar
  17. Davis AP (1993) Cholaphanes et al.; steroids as structural components in molecular engineering. Chem Soc Rev 22(4):243–253. CrossRefGoogle Scholar
  18. de Miranda P, Blum MR (1983) Pharmacokinetics of acyclovir after intravenous and oral administration. J Antimicrob Chemother 12 Suppl B:29–37CrossRefPubMedGoogle Scholar
  19. de Miranda P, Krasny HC, Page DA, Elion GB (1981) The disposition of acyclovir in different species. J Pharmacol Exp Ther 219(2):309–315PubMedGoogle Scholar
  20. de Miranda P, Krasny HC, Page DA, Elion GB (1982) Species differences in the disposition of acyclovir. Am J Med 73(1A):31–35CrossRefPubMedGoogle Scholar
  21. Duane WC, Xiong W, Wolvers J (2007) Effects of bile acids on expression of the human apical sodium dependent bile acid transporter gene. Biochim Biophys Acta 1771(11):1380–1388. CrossRefPubMedGoogle Scholar
  22. Elnaggar YS (2015) Multifaceted applications of bile salts in pharmacy: an emphasis on nanomedicine. Int J Nanomed 10:3955–3971. CrossRefGoogle Scholar
  23. Enhsen A, Kramer W, Wess G (1998) Bile acids in drug discovery. Drug Discov Today 3(9):409–418CrossRefGoogle Scholar
  24. Guo A, Hu P, Balimane PV, Leibach FH, Sinko PJ (1999) Interactions of a nonpeptidic drug, valacyclovir, with the human intestinal peptide transporter (hPEPT1) expressed in a mammalian cell line. J Pharmacol Exp Ther 289(1):448–454PubMedGoogle Scholar
  25. Gupta S, Agarwal A, Gupta NK, Saraogi G, Agrawal H, Agrawal GP (2013) Galactose decorated PLGA nanoparticles for hepatic delivery of acyclovir. Drug Dev Ind Pharm 39(12):1866–1873. CrossRefPubMedGoogle Scholar
  26. Han H, de Vrueh RL, Rhie JK, Covitz KM, Smith PL, Lee CP, Oh DM, Sadee W, Amidon GL (1998) 5′-Amino acid esters of antiviral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1 peptide transporter. Pharm Res 15(8):1154–1159CrossRefPubMedGoogle Scholar
  27. Hofmann A (1988) In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritz DA (eds) The liver: biology and pathobiology. Raven Press, New York, pp 553–572Google Scholar
  28. Johnston C, Saracino M, Kuntz S, Magaret A, Selke S, Huang ML, Schiffer JT, Koelle DM, Corey L, Wald A (2012) Standard-dose and high-dose daily antiviral therapy for short episodes of genital HSV-2 reactivation: three randomised, open-label, cross-over trials. Lancet 379(9816):641–647. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kagedahl M, Swaan PW, Redemann CT, Tang M, Craik CS, Szoka FC Jr, Oie S (1997) Use of the intestinal bile acid transporter for the uptake of cholic acid conjugates with HIV-1 protease inhibitory activity. Pharm Res 14(2):176–180CrossRefPubMedGoogle Scholar
  30. Karaman R, Dajani KK, Qtait A, Khamis M (2012) Prodrugs of acyclovir—a computational approach. Chem Biol Drug Des 79(5):819–834. CrossRefPubMedGoogle Scholar
  31. Katragadda S, Jain R, Kwatra D, Hariharan S, Mitra AK (2008) Pharmacokinetics of amino acid ester prodrugs of acyclovir after oral administration: interaction with the transporters on Caco-2 cells. Int J Pharm 362(1–2):93–101. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kim DC, Harrison AW, Ruwart MJ, Wilkinson KF, Fisher JF, Hidalgo IJ, Borchardt RT (1993) Evaluation of the bile acid transporter in enhancing intestinal permeability to renin-inhibitory peptides. J Drug Target 1(4):347–359. CrossRefPubMedGoogle Scholar
  33. Kimberlin DW, Whitley RJ (2007) Antiviral therapy of HSV-1 and -2. In: Arvin A, Campadelli-Fiume G, Mocarski E et al (eds) Human herpesviruses: biology, therapy, and immunoprophylaxis. Cambridge University Press, CambridgeGoogle Scholar
  34. Klysik K, Pietraszek A, Karewicz A, Nowakowska M (2018) Acyclovir in the treatment of herpes viruses—a review. Curr Med Chem. PubMedCrossRefGoogle Scholar
  35. Kramer W, Glombik H (2006) Bile acid reabsorption inhibitors (BARI): novel hypolipidemic drugs. Curr Med Chem 13(9):997–1016CrossRefPubMedGoogle Scholar
  36. Kramer W, Wess G (1996) Bile acid transport systems as pharmaceutical targets. Eur J Clin Invest 26(9):715–732CrossRefPubMedGoogle Scholar
  37. Kramer W, Wess G, Schubert G, Bickel M, Girbig F, Gutjahr U, Kowalewski S, Baringhaus KH, Enhsen A, Glombik H et al (1992) Liver-specific drug targeting by coupling to bile acids. J Biol Chem 267(26):18598–18604PubMedGoogle Scholar
  38. Kramer W, Wess G, Enhsen A, Falk E, Hoffmann A, Neckermann G, Schubert G, Urmann M (1997) Modified bile acids as carriers for peptides and drugs. J Control Release 46(1):17–30. CrossRefGoogle Scholar
  39. Lack L (1979) Properties and biological significance of the ileal bile salt transport system. Environ Health Perspect 33:79–89CrossRefPubMedPubMedCentralGoogle Scholar
  40. Li Y, Dias JR (1997) Dimeric and oligomeric steroids. Chem Rev 97(1):283–304CrossRefPubMedGoogle Scholar
  41. Liang R, Fei YJ, Prasad PD, Ramamoorthy S, Han H, Yang-Feng TL, Hediger MA, Ganapathy V, Leibach FH (1995) Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization. J Biol Chem 270(12):6456–6463CrossRefPubMedGoogle Scholar
  42. Longerich T, Eisenbach C, Penzel R, Kremer T, Flechtenmacher C, Helmke B, Encke J, Kraus T, Schirmacher P (2005) Recurrent herpes simplex virus hepatitis after liver retransplantation despite acyclovir therapy. Liver Transpl 11(10):1289–1294. CrossRefPubMedGoogle Scholar
  43. Love MW, Dawson PA (1998) New insights into bile acid transport. Curr Opin Lipidol 9(3):225–229CrossRefPubMedGoogle Scholar
  44. Miller G, Lipman M (1973) Release of infectious Epstein–Barr virus by transformed marmoset leukocytes. Proc Natl Acad Sci USA 70(1):190–194CrossRefPubMedGoogle Scholar
  45. Mukhopadhyay S, Maitra U (2004) Chemistry and biology of bile acids. Curr Sci 87(12):1666–1683Google Scholar
  46. Norvell JP, Blei AT, Jovanovic BD, Levitsky J (2007) Herpes simplex virus hepatitis: an analysis of the published literature and institutional cases. Liver Transpl 13(10):1428–1434. CrossRefPubMedGoogle Scholar
  47. Park G-B, Shao Z, Mitra AK (1992) Acyclovir permeation enhancement across intestinal and nasal mucosae by bile salt-acylcarnitine mixed micelles. Pharm Res 9(10):1262–1267. CrossRefPubMedGoogle Scholar
  48. Petzinger E (1994) Transport of organic anions in the liver. An update on bile acid, fatty acid, monocarboxylate, anionic amino acid, cholephilic organic anion, and anionic drug transport. Rev Physiol Biochem Pharmacol 123:47–211CrossRefPubMedGoogle Scholar
  49. Pyles RB (2001) The association of herpes simplex virus and Alzheimer’s disease: a potential synthesis of genetic and environmental factors. Herpes J IHMF 8(3):64–68Google Scholar
  50. Rais R, Fletcher S, Polli JE (2011) Synthesis and in vitro evaluation of gabapentin prodrugs that target the human apical sodium-dependent bile acid transporter (hASBT). J Pharm Sci 100(3):1184–1195. CrossRefPubMedGoogle Scholar
  51. Santos CR, Capela R, Pereira CSGP, Valente E, Gouveia L, Pannecouque C, De Clercq E, Moreira R, Gomes P (2009) Structure–activity relationships for dipeptide prodrugs of acyclovir: implications for prodrug design. Eur J Med Chem 44(6):2339–2346. CrossRefPubMedGoogle Scholar
  52. Shao Z, Hoffman AJ, Mitra AK (1994) Biodegradation characteristics of acyclovir 2′-esters by respiratory carboxylesterases: implications in prodrug design for intranasal and pulmonary drug delivery. Int J Pharm 112(2):181CrossRefGoogle Scholar
  53. Sievanen E (2007) Exploitation of bile acid transport systems in prodrug design. Molecules 12(8):1859–1889CrossRefPubMedGoogle Scholar
  54. Soul-Lawton J, Seaber E, On N, Wootton R, Rolan P, Posner J (1995) Absolute bioavailability and metabolic disposition of valaciclovir, the L-valyl ester of acyclovir, following oral administration to humans. Antimicrob Agents Chemother 39(12):2759–2764CrossRefPubMedPubMedCentralGoogle Scholar
  55. Staels B, Fonseca VA (2009) Bile acids and metabolic regulation: mechanisms and clinical responses to bile acid sequestration. Diabetes Care 32(Suppl 2):S237–S245. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Stedronsky ER (1994) Interaction of bile acids and cholesterol with non-systemic agents having hypocholesterolemic properties. Biochim Biophys Acta 1210(3):255–287CrossRefPubMedGoogle Scholar
  57. Stojančević M, Pavlović N, Goločorbin-Kon S, Mikov M (2013) Application of bile acids in drug formulation and delivery. Front Life Sci 7(3–4):112–122. CrossRefGoogle Scholar
  58. St-Pierre MV, Kullak-Ublick GA, Hagenbuch B, Meier PJ (2001) Transport of bile acids in hepatic and non-hepatic tissues. J Exp Biol 204(Pt 10):1673–1686PubMedGoogle Scholar
  59. Swaan PW, Hillgren KM, Szoka FC, Øie S (1997a) Enhanced transepithelial transport of peptides by conjugation to cholic acid. Bioconj Chem 8(4):520–525. CrossRefGoogle Scholar
  60. Swaan PW, Szoka FC Jr, Oie S (1997b) Molecular modeling of the intestinal bile acid carrier: a comparative molecular field analysis study. J Comput Aided Mol Des 11(6):581–588CrossRefPubMedGoogle Scholar
  61. Tamminen J, Kolehmainen E (2001) Bile acids as building blocks of supramolecular hosts. Molecules 6(1):21CrossRefGoogle Scholar
  62. Thamotharan M, Lombardo YB, Bawani SZ, Adibi SA (1997) An active mechanism for completion of the final stage of protein degradation in the liver, lysosomal transport of dipeptides. J Biol Chem 272(18):11786–11790CrossRefPubMedGoogle Scholar
  63. Tolle-Sander S, Lentz KA, Maeda DY, Coop A, Polli JE (2004) Increased acyclovir oral bioavailability via a bile acid conjugate. Mol Pharm 1(1):40–48CrossRefPubMedGoogle Scholar
  64. Turley DS, Dietschy JM (1988) In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritz DS (eds) In the liver: biology and pathobiology. Raven Press, New York, pp 617–641Google Scholar
  65. Virtanen E, Kolehmainen E (2004) Use of bile acids in pharmacological and supramolecular applications. Eur J Org Chem 16:3385–3399. CrossRefGoogle Scholar
  66. Vivian D, Polli JE (2014) Synthesis and in vitro evaluation of bile acid prodrugs of floxuridine to target the liver. Int J Pharm 475(1–2):597–604. CrossRefPubMedPubMedCentralGoogle Scholar
  67. Wallimann P, Marti T, Fürer A, Diederich F (1997) Steroids in molecular recognition. Chem Rev 97(5):1567–1608. CrossRefPubMedGoogle Scholar
  68. Wang J, Yan X, Lu R, Meng X, Nie G (2017) Peptide transporter 1 (PepT1) in fish: a review. Aquac Fish 2(5):193–206. CrossRefGoogle Scholar
  69. Yang C, Tirucherai GS, Mitra AK (2001) Prodrug based optimal drug delivery via membrane transporter/receptor. Expert Opin Biol Ther 1(2):159–175. CrossRefPubMedGoogle Scholar
  70. Zhang D, Li D, Shang L, He Z, Sun J (2016) Transporter-targeted cholic acid-cytarabine conjugates for improved oral absorption. Int J Pharm 511(1):161–169. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Radoslav L. Chayrov
    • 1
  • Evgenios K. Stylos
    • 5
    • 6
  • Maria V. Chatziathanasiadou
    • 5
  • Kiril N. Chuchkov
    • 1
  • Aleksandra I. Tencheva
    • 1
  • Androniki D. Kostagianni
    • 5
  • Tsenka S. Milkova
    • 1
  • Assia L. Angelova
    • 2
    • 3
  • Angel S. Galabov
    • 3
  • Stoyan A. Shishkov
    • 4
  • Daniel G. Todorov
    • 4
  • Andreas G. Tzakos
    • 5
    Email author
  • Ivanka G. Stankova
    • 1
    Email author
  1. 1.Department of ChemistrySouth-West University “Neofit Rilski”BlagoevgradBulgaria
  2. 2.Department of Tumor VirologyGerman Cancer Research CenterHeidelbergGermany
  3. 3.Department of Virology, Institute of MicrobiologyBulgarian Academy of SciencesSofiaBulgaria
  4. 4.Laboratory of VirologySofia University “St. Kliment Ohridski”SofiaBulgaria
  5. 5.Section of Organic Chemistry and Biochemistry, Department of ChemistryUniversity of IoanninaIoanninaGreece
  6. 6.Biotechnology Laboratory, Department of Biological Applications and TechnologyUniversity of IoanninaIoanninaGreece

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