Antiviral Research 125 (2016) 58e62
Contents lists available at ScienceDirect
Antiviral Research journal homepage: www.elsevier.com/locate/antiviral
Short communication
Respiratory syncytial virus infection in macaques is not suppressed by intranasal sprays of pyrimidine biosynthesis inhibitors ment Grandin a, b, c, 1, Marianne-Lucas Hourani b, c, 1, Yves L. Janin d, e, Cle le ne Munier-Lehmann d, e, Adeline Paturet a, Fabrice Taborik a, Daniel Dauzonne f, g, h, He i de ric Tangy b, c, **, Pierre-Olivier Vidalain b, c, * Astrid Vabret , Hugues Contamin a, ***, Fre a
Cynbiose SA, Marcy-l'Etoile, France Institut Pasteur, Unit e de G enomique Virale et Vaccination, Paris, France CNRS, UMR3569, Paris, France d Institut Pasteur, Unit e de Chimie et Biocatalyse, Paris, France e CNRS, UMR3523, Paris, France f Institut Curie, Centre de Recherche, Paris, France g CNRS, UMR3666, Paris, France h INSERM, U1143, Paris, France i Universit e de Caen-Basse-Normandie, EA 4655-U2RM, Laboratoire de Virologie, CHU de Caen, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 June 2015 Received in revised form 16 October 2015 Accepted 14 November 2015 Available online 22 November 2015
There is imperious need for efficient therapies against ubiquitous and life-threatening respiratory viruses, foremost among them being the human respiratory syncytial virus (hRSV). Several research groups who performed functional screens for broad-spectrum antivirals identified compounds targeting the de novo pyrimidine biosynthesis pathway. Despite their strong antiviral activity in vitro, whether such antimetabolites are effective in vivo remains highly controversial. Here, we evaluated two potent pyrimidine biosynthesis inhibitors developed in our laboratory, IPPA17-A04 and GAC50, in a model of mild hRSV-infection in cynomolgus macaques. In this model, hRSV replication is restricted to the epithelium of the upper respiratory tract, and is compatible with a topical treatment by intranasal sprays. The local administration of palivizumab, a neutralizing anti-hRSV antibody used in clinics, significantly reduced virus replication. In contrast, pyrimidine biosynthesis inhibitors did not show any inhibitory effect on hRSV growth when delivered topically as experimented in our model. Our results should help to better define the potential applications of this class of antimetabolites in the treatment of viral infections. © 2015 Elsevier B.V. All rights reserved.
Keywords: Cynomolgus macaques Respiratory syncytial virus Pyrimidine biosynthesis inhibitors Antiviral compounds Dihydroorotate dehydrogenase
Human respiratory syncytial virus (hRSV) is a negative-strand RNA virus of major incidence worldwide that is responsible for
Abbreviations: hRSV, human respiratory syncytial virus; DHODH, dihydroorotate dehydrogenase; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline; RT-qPCR, reverse transcription and quantitative real-time polymerase chain reaction. * Corresponding author. Present address: Laboratoire de Chimie et Biochimie Paris Descartes, CNRS, Pharmacologiques et Toxicologiques, Team CBNIT, Universite UMR8601, Paris, France. de Ge nomique Virale et Vacci** Corresponding author. Institut Pasteur, Unite nation, CNRS, UMR3569, Paris, France. *** Corresponding author. Cynbiose SA, Marcy-l'Etoile, France. E-mail addresses: [email protected] (H. Contamin), frederic. [email protected] (F. Tangy), [email protected] (P.-O. Vidalain). 1 Equally contributors. http://dx.doi.org/10.1016/j.antiviral.2015.11.006 0166-3542/© 2015 Elsevier B.V. All rights reserved.
life-threatening respiratory infections in newborns, elderly and immunocompromised people (Borchers et al., 2013; Collins and Melero, 2011). Despite persistent efforts, there is no effective vaccine or specific therapy available against hRSV, with the noticeable exception of palivizumab, a humanized monoclonal antibody directed against the F glycoprotein of the virus. Palivizumab is administered by intramuscular injection to high-risk infants during the epidemic season as a prophylactic treatment, but its use is limited by a low benefit-cost ratio (Collins and Melero, 2011). Several compounds inhibiting de novo pyrimidine biosynthesis were recently characterized as broad-spectrum antivirals (Bonavia et al., 2011; Hoffmann et al., 2011; Lucas-Hourani et al., 2013, 2015; Marschall et al., 2013; Munier-Lehmann et al., 2015; Ortiz-Riano et al., 2014; Smee et al., 2012; Wang et al., 2011). Most of these molecules target the fourth enzyme of this metabolic pathway,
C. Grandin et al. / Antiviral Research 125 (2016) 58e62
called DHODH (dihydroorotate dehydrogenase), and were shown to inhibit in vitro a large panel of RNA and DNA viruses, including hRSV (Bonavia et al., 2011). Pyrimidine nucleosides are essential precursors for nucleic acid synthesis, they act as intermediates in multiple metabolic pathways, and depleting cells of pyrimidine nucleosides can boost the innate immune response to viruses (Munier-Lehmann et al., 2013). Altogether, this explains the antiviral activity of pyrimidine biosynthesis inhibitors. Despite promising results in vitro, the antiviral efficacy of these antimetabolites in vivo remains controversial. Various pyrimidine
59
biosynthesis inhibitors failed to reduce the replication of dengue virus, influenza virus or hRSV in experimentally-infected rodents (Bonavia et al., 2011; Smee et al., 2012; Wang et al., 2011), although one compound efficiently inhibited cytomegalovirus replication in mice (Marschall et al., 2013). Controversial results were also obtained with leflunomide, a drug that inhibits both tyrosine kinases and de novo pyrimidine biosynthesis, in rodent models of hRSV infection (Cox Dunn et al., 2011; Davis et al., 2006). Thus, additional experimentations are needed to determine if pyrimidine biosynthesis inhibitors are effective in vivo against virus infections, and
Fig. 1. In vitro inhibition of hRSV growth by IPPA17-A04 and GAC50. (A) Chemical structures of GAC50 and IPPA17-A04. (B and C) hRSV growth was determined in Hep2 cells infected with hRSV (strain Long; MOI ¼ 0.1), and cultured with DMSO alone, or increasing concentrations of IPPA17-A04 (B) or GAC50 (C). Viral RNA copies were determined after 72 h of culture by RT-qPCR as previously described (Grandin et al., 2015). Data show means ± SD of one representative experiment performed in triplicate. ** indicates p-values < 0.01 as determined by standard t-test (D) Viral protein expression in hRSV-infected LLC-MK2 cells (MOI ¼ 0.1) when treated with IPPA17-A04, GAC50 or left untreated (DMSO alone). hRSV-F protein expression was determined by immunostaining using a mouse monoclonal antibody (MAB8599; clone 131-2A).
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C. Grandin et al. / Antiviral Research 125 (2016) 58e62
hRSV in particular. We recently set up a model of hRSV infection in cynomolgus macaques where the virus is administered by the intranasal route and replicates for less than 15 days in the upper respiratory tract without inducing any pathology (Grandin et al., 2015). We took advantage of this mild-infection model to evaluate the antiviral
potential of IPPA17-A04 and GAC50 (Fig. 1A), two optimized pyrimidine biosynthesis inhibitors developed in our laboratory (Lucas-Hourani et al., 2013, 2015; Munier-Lehmann et al., 2015). This model was selected because GAC50 is only active on primate cells (data not shown). Furthermore, the antiviral activity of pyrimidine biosynthesis inhibitors has never been evaluated in
Fig. 2. Evaluation of palivizumab, IPPA17-A04 and GAC50 antiviral activity in the hRSV-infection macaque model. (A) Timeline of infection, samples collection and treatments. Oneyear old animals were infected intranasally with 5.10þ4 TCID50 of hRSV, and treated with palivizumab, IPPA17-A04 or GAC50 from day 2e9 post-infection. Nasal swabs were collected on all animals at indicated time-points. (BeE) Viral RNA copies detected in nasal swab specimens from control animals (B), or animals treated intranasally with palivizumab (C), IPPA17-A04 (D), or GAC50 (E). hRSV RNA copies in nasal swab specimens were quantified by RT-qPCR, and normalized relative to cell count in each sample as previously described (Grandin et al., 2015). Results correspond to the average of viral RNA copies (log values) from left and right nostrils of each animal. Control animals in (B) were left untreated (55 and 56) or treated with a PBS þ 10% Kolliphor solution (57, 58, and 59), which is used as a carrier for IPPA17-A04 and GAC50. (F) Means of viral RNA copies (log values) for each group of animals. The control curve corresponds to the average of viral RNA copies from all control animals (55e59). (G) Nasal swab specimens collected at day 12 from animals left untreated (55e59) or treated with IPPA17-A04 (60e64) or GAC50 (65e69) were clarified by centrifugation, and then tested at a 1/8 dilution for their antiviral activity in a luciferase-based virus growth assay (Lucas-Hourani et al., 2014). This assay relies on HEK-293T cell cultures infected by a recombinant strain of measles virus expressing luciferase, and is highly sensitive to pyrimidine biosynthesis inhibitors. Culture medium was supplemented or not with uridine at 20 mg/ml.
C. Grandin et al. / Antiviral Research 125 (2016) 58e62
primates before and in this model, it was possible to deliver the drug topically by intranasal sprays at the site of virus replication. Prior to in vivo experimentations, compounds were assessed in vitro for hRSV growth inhibition, cytoxicity and metabolic stability. First, IPPA17-A04 and GAC50 were shown to block hRSV replication in human Hep2 cells (Fig. 1B and C), and IC50s were estimated to 5 nM and 25 mM, respectively. The inhibition of hRSV growth was confirmed in the macaque cell line LLC-MK2 by anti-F immunostaining (Fig. 1D). In parallel, the half maximal cytotoxic concentration (CC50) of the compounds was determined using a commercial cell viability assay (CellTiter-Glo reagent; Promega). CC50s of IPPA17-A04 and GAC50 were >500 and 250 mM, respectively, both in Hep2 and LLC-MK2 cells. Metabolic stabilities of the compounds were determined on human liver microsomes (TechMedILL, Illkirch). The half-life of IPPA17-A04 was good (42 min), but GAC50 was rapidly metabolized (11 min). Because IPPA17-A04 showed a higher activity and better metabolic stability than GAC50, we documented its general toxicity and pharmacokinetic in macaques. IPPA17-A04 was injected intravenously to three animals during 6 days at 20 mg/kg/day, and plasmatic concentrations were measured by mass spectrometry at seven timepoints after the first and the last injection (Oroxcell, Romainville). The mean apparent terminal half-life of IPPA17-A04 in plasma was 1.2 ± 0.13 h after the first injection, but increased to 10.1 ± 3.11 h for the last injection, suggesting an accumulation of the molecule in tissues after multiple daily injections. Macroscopic and histopathological studies of the animal tissues after the 6th injection showed no sign of toxicity (Novotec, Lyon). Since IPPA17-A04, our most active and stable molecule, was not toxic for macaques, the two compounds were evaluated for their antiviral activity in vivo. Four groups of five animals were infected with hRSV as previously described (Grandin et al., 2015), and treated from day 2e9 post-infection with IPPA17-04, GAC50, palivizumab, or were left untreated (Fig. 2A). IPPA17-A04 and GAC50 were formulated in PBS þ10% Kolliphor EL at 1 mg/ml, below their limits of solubility (respectively 1.7 and 1.47 mg/ml; TechMedILL), whereas palizivumab was resuspended in PBS at 2.5 mg/ml. Treatments were administered topically in the upper respiratory tract with an intranasal sprayer (MicroSprayer Aerosolizer e Model IA-1B; PennCentury) to target the virus replication site. Animals received once a day in each nostril 1.25 mg of palivizumab, or 0.5 mg of IPPA17-A04 or GAC50. This approximately corresponds to 0.2 mg/ kg/day, a regimen comparable to intranasal doses previously administered in human when evaluating influenza virus or rhinovirus inhibitors with in vitro antiviral activities in the nM to mM range (Calfee et al., 1999; Hayden et al., 1992, 2003; Matsumoto et al., 1999). Three of the five untreated animals received once a day a matching volume of the PBS þ10% Kolliphor EL solution used to dissolve IPPA17-A04 and GAC50. The two other control animals received no treatment at all. Nasal swabs were collected from both left and right nostrils from day 5 before the infection up to day 29 post-infection. Then, total RNA was extracted from nasal swabs, and the number of hRSV RNA copies for 10þ3 cells determined by RTqPCR (Grandin et al., 2015). This protocol was approved by the Animal Care and Use Committee of VetAgro Sup. In this model, the initial inoculum is detected at day 1 postinfection, and is cleared in about 3 days (Grandin et al., 2015). The rebound in virus load detected at day 4e5 unambiguously demonstrates viral replication. As shown in Fig. 2B, numbers of viral RNA copies were virtually similar at the peak of infection in animals left untreated (55 and 56) or treated with a PBS þ 10% Kolliphor solution (57, 58, and 59). Viral RNA copies in palivizumabtreated animals were found 2.7 to 29 times lower than in controls (Fig. 2C and F), and inhibition was highly significant (two-way ANOVA; p-value ¼ 0.0036). This prooved palivizumab efficacy, and
61
also demonstrated that the intranasal spray efficiently delivered the antiviral molecule at the site of virus replication. In contrast, IPPA17-A04 or GAC50 did not show any inhibitory effect on hRSV growth between day 4 and 11 (Fig. 2DeF), and the lower viral load detected at day 3 in compound-treated animals was not statistically significant. In contrast, IPPA17-A04 treatment significantly increased viral loads at the peak of replication from day 5e9 (twoway ANOVA; p-value ¼ 0.0494), suggesting some proviral effects. In conclusion, IPPA17-A04 and GAC50 failed to prevent hRSV growth in macaques. We cannot exclude that tested compounds had no effect because they were quickly metabolized. However, some antiviral activity was still detected with a sensitive virus replication assay (Lucas-Hourani et al., 2014) in nasal swab specimens collected at day 12, i.e. up to 3 days after the last administration (Fig. 2G). This activity was reversed by the addition of uridine, supporting the presence of pyrimidine biosynthesis inhibitors in nasal swab specimens. It could be also that delivered doses were too low or compounds were not absorbed by the epithelial tissue. However, the proviral activity of IPPA17-A04 suggests that this was not the case, at least for this molecule. Altogether, our observations are consistent with the negative results reported in hRSV-infected rodents treated with pyrimidine biosynthesis inhibitors by oral gavage (Bonavia et al., 2011; Davis et al., 2006). How can we explain this setback? Besides de novo pyrimidine biosynthesis, cells can acquire pyrimidine nucleosides by the salvage of nucleobases from catabolic pathways and uptakes from the extracellular pool (Munier-Lehmann et al., 2013). Epithelial cells from the respiratory tract could have their needs in pyrimidine nucleosides fulfilled by these two alternative mechanisms if their metabolic activity is limited. This hypothesis should be supported by in situ quantification of pyrimidine levels in treated macaques, something we have not investigated yet. Finally, results obtained with IPPA17-A04 suggest that in vivo, pyrimidine biosynthesis inhibitors can have proviral effects. One hypothesis is that blocking this metabolic pathway could interfere with cellmediated immunity. Indeed, lymphocytes have important needs in nucleosides when proliferating, and inhibitors of de novo pyrimidine biosynthesis can block their expansion both in vitro and in vivo (Munier-Lehmann et al., 2013). In conclusion, our results challenge the development of pyrimidine biosynthesis inhibitors as a treatment against hRSV infection. Acknowledgments This work was supported by the National Research Agency ^le, the (ANR-2011-RPIB-006-01, Program STING 2.0), Lyonbiopo Institut Carnot Pasteur MI (Program STING), the Institut Pasteur, the et de la Recherche Me dicale, and the Institut National de la Sante Centre National de la Recherche Scientifique. CG was supported by a CIFRE fellowship (Conventions Industrielles de Formation par la Recherche, Association Nationale de la Recherche et de la Technologie; 2011/1187). We thank Dr. Jean-François Bureau for his advice on statistical analyses. We thank Dr. Mustapha Si-Tahar and Dr. Nathalie Heuze-Vourc'h for their technical advices on aerosolization technics. References Bonavia, A., Franti, M., Pusateri Keaney, E., Kuhen, K., Seepersaud, M., Radetich, B., Shao, J., Honda, A., Dewhurst, J., Balabanis, K., Monroe, J., Wolff, K., Osborne, C., Lanieri, L., Hoffmaster, K., Amin, J., Markovits, J., Broome, M., Skuba, E., CornellaTaracido, I., Joberty, G., Bouwmeester, T., Hamann, L., Tallarico, J.A., Tommasi, R., Compton, T., Bushell, S.M., 2011. Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV). Proc. Natl. Acad. Sci. U. S. A. 108, 6739e6744. Borchers, A.T., Chang, C., Gershwin, M.E., Gershwin, L.J., 2013. Respiratory syncytial
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virus - a comprehensive review. Clin. Rev. Allergy Immunol. 45, 331e379. Calfee, D.P., Peng, A.W., Hussey, E.K., Lobo, M., Hayden, F.G., 1999. Safety and efficacy of once daily intranasal zanamivir in preventing experimental human influenza A infection. Antivir. Ther. 4, 143e149. Collins, P.L., Melero, J.A., 2011. Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. Virus Res. 162, 80e99. Cox Dunn, M.C., Knight, D.A., Waldman, W.J., 2011. Inhibition of respiratory syncitial virus in vitro and in vivo by the immunosuppressive agent leflunomide. Antivir. Ther. 16, 309e317. Davis, I.C., Lazarowski, E.R., Hickman-Davis, J.M., Fortenberry, J.A., Chen, F.P., Zhao, X., Sorscher, E., Graves, L.M., Sullender, W.M., Matalon, S., 2006. Leflunomide prevents alveolar fluid clearance inhibition by respiratory syncytial virus. Am. J. Respir. Crit. Care Med. 173, 673e682. Grandin, C., Lucas-Hourani, M., Clavel, M., Taborik, F., Vabret, A., Tangy, F., Contamin, H., Vidalain, P.O., 2015. Evidence for an intranasal immune response to human respiratory syncytial virus infection in cynomolgus macaques. J. Gen. Virol. 96, 782e792. Hayden, F.G., Andries, K., Janssen, P.A., 1992. Safety and efficacy of intranasal pirodavir (R77975) in experimental rhinovirus infection. Antimicrob. Agents Chemother. 36, 727e732. Hayden, F.G., Turner, R.B., Gwaltney, J.M., Chi-Burris, K., Gersten, M., Hsyu, P., Patick, A.K., Smith 3rd, G.J., Zalman, L.S., 2003. Phase II, randomized, doubleblind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers. Antimicrob. Agents Chemother. 47, 3907e3916. Hoffmann, H.H., Kunz, A., Simon, V.A., Palese, P., Shaw, M.L., 2011. Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 108, 5777e5782. Lucas-Hourani, M., Dauzonne, D., Jorda, P., Cousin, G., Lupan, A., Helynck, O., Caignard, G., Janvier, G., Andre-Leroux, G., Khiar, S., Escriou, N., Despres, P., Jacob, Y., Munier-Lehmann, H., Tangy, F., Vidalain, P.O., 2013. Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity. PLoS Pathog. 9, e1003678. Lucas-Hourani, M., Munier-Lehmann, H., El Mazouni, F., Malmquist, N.A., Harpon, J.,
Coutant, E.P., Guillou, S., Helynck, O., Noel, A., Scherf, A., Phillips, M.A., Tangy, F., Vidalain, P.O., Janin, Y.L., 2015. Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines inhibitors of human dihydroorotate dehydrogenase (DHODH). J. Med. Chem. 58, 5579e5598. Lucas-Hourani, M., Munier-Lehmann, H., Helynck, O., Komarova, A., Despres, P., Tangy, F., Vidalain, P.O., 2014. High-throughput screening for broad-spectrum chemical inhibitors of RNA viruses. J. Vis. Exp. 87. Marschall, M., Niemann, I., Kosulin, K., Bootz, A., Wagner, S., Dobner, T., Herz, T., Kramer, B., Leban, J., Vitt, D., Stamminger, T., Hutterer, C., Strobl, S., 2013. Assessment of drug candidates for broad-spectrum antiviral therapy targeting cellular pyrimidine biosynthesis. Antivir. Res. 100, 640e648. Matsumoto, K., Ogawa, N., Nerome, K., Numazaki, Y., Kawakami, Y., Shirato, K., Arakawa, M., Kudoh, S., Shimokata, K., Nakajima, S., Yamakido, M., Kashiwagi, S., Nagatake, T., 1999. Safety and efficacy of the neuraminidase inhibitor zanamivir in treating influenza virus infection in adults: results from Japan. GG167 Group. Antivir. Ther. 4, 61e68. Munier-Lehmann, H., Lucas-Hourani, M., Guillou, S., Helynck, O., Zanghi, G., Noel, A., Tangy, F., Vidalain, P.O., Janin, Y.L., 2015. Original 2-(3-Alkoxy-1H-pyrazol-1-yl) pyrimidine Derivatives as inhibitors of human dihydroorotate dehydrogenase (DHODH). J. Med. Chem. 58, 860e877. Munier-Lehmann, H., Vidalain, P.O., Tangy, F., Janin, Y.L., 2013. On dihydroorotate dehydrogenases and their inhibitors and uses. J. Med. Chem. 56, 3148e3167. Ortiz-Riano, E., Ngo, N., Devito, S., Eggink, D., Munger, J., Shaw, M.L., de la Torre, J.C., Martinez-Sobrido, L., 2014. Inhibition of arenavirus by A3, a pyrimidine biosynthesis inhibitor. J. Virol. 88, 878e889. Smee, D.F., Hurst, B.L., Day, C.W., 2012. D282, a non-nucleoside inhibitor of influenza virus infection that interferes with de novo pyrimidine biosynthesis. Antivir. Chem. Chemother. 22, 263e272. Wang, Q.Y., Bushell, S., Qing, M., Xu, H.Y., Bonavia, A., Nunes, S., Zhou, J., Poh, M.K., Florez de Sessions, P., Niyomrattanakit, P., Dong, H., Hoffmaster, K., Goh, A., Nilar, S., Schul, W., Jones, S., Kramer, L., Compton, T., Shi, P.Y., 2011. Inhibition of dengue virus through suppression of host pyrimidine biosynthesis. J. Virol. 85, 6548e6556.
Contents lists available at ScienceDirect
Antiviral Research journal homepage: www.elsevier.com/locate/antiviral
Short communication
Respiratory syncytial virus infection in macaques is not suppressed by intranasal sprays of pyrimidine biosynthesis inhibitors ment Grandin a, b, c, 1, Marianne-Lucas Hourani b, c, 1, Yves L. Janin d, e, Cle le ne Munier-Lehmann d, e, Adeline Paturet a, Fabrice Taborik a, Daniel Dauzonne f, g, h, He i de ric Tangy b, c, **, Pierre-Olivier Vidalain b, c, * Astrid Vabret , Hugues Contamin a, ***, Fre a
Cynbiose SA, Marcy-l'Etoile, France Institut Pasteur, Unit e de G enomique Virale et Vaccination, Paris, France CNRS, UMR3569, Paris, France d Institut Pasteur, Unit e de Chimie et Biocatalyse, Paris, France e CNRS, UMR3523, Paris, France f Institut Curie, Centre de Recherche, Paris, France g CNRS, UMR3666, Paris, France h INSERM, U1143, Paris, France i Universit e de Caen-Basse-Normandie, EA 4655-U2RM, Laboratoire de Virologie, CHU de Caen, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 June 2015 Received in revised form 16 October 2015 Accepted 14 November 2015 Available online 22 November 2015
There is imperious need for efficient therapies against ubiquitous and life-threatening respiratory viruses, foremost among them being the human respiratory syncytial virus (hRSV). Several research groups who performed functional screens for broad-spectrum antivirals identified compounds targeting the de novo pyrimidine biosynthesis pathway. Despite their strong antiviral activity in vitro, whether such antimetabolites are effective in vivo remains highly controversial. Here, we evaluated two potent pyrimidine biosynthesis inhibitors developed in our laboratory, IPPA17-A04 and GAC50, in a model of mild hRSV-infection in cynomolgus macaques. In this model, hRSV replication is restricted to the epithelium of the upper respiratory tract, and is compatible with a topical treatment by intranasal sprays. The local administration of palivizumab, a neutralizing anti-hRSV antibody used in clinics, significantly reduced virus replication. In contrast, pyrimidine biosynthesis inhibitors did not show any inhibitory effect on hRSV growth when delivered topically as experimented in our model. Our results should help to better define the potential applications of this class of antimetabolites in the treatment of viral infections. © 2015 Elsevier B.V. All rights reserved.
Keywords: Cynomolgus macaques Respiratory syncytial virus Pyrimidine biosynthesis inhibitors Antiviral compounds Dihydroorotate dehydrogenase
Human respiratory syncytial virus (hRSV) is a negative-strand RNA virus of major incidence worldwide that is responsible for
Abbreviations: hRSV, human respiratory syncytial virus; DHODH, dihydroorotate dehydrogenase; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline; RT-qPCR, reverse transcription and quantitative real-time polymerase chain reaction. * Corresponding author. Present address: Laboratoire de Chimie et Biochimie Paris Descartes, CNRS, Pharmacologiques et Toxicologiques, Team CBNIT, Universite UMR8601, Paris, France. de Ge nomique Virale et Vacci** Corresponding author. Institut Pasteur, Unite nation, CNRS, UMR3569, Paris, France. *** Corresponding author. Cynbiose SA, Marcy-l'Etoile, France. E-mail addresses: [email protected] (H. Contamin), frederic. [email protected] (F. Tangy), [email protected] (P.-O. Vidalain). 1 Equally contributors. http://dx.doi.org/10.1016/j.antiviral.2015.11.006 0166-3542/© 2015 Elsevier B.V. All rights reserved.
life-threatening respiratory infections in newborns, elderly and immunocompromised people (Borchers et al., 2013; Collins and Melero, 2011). Despite persistent efforts, there is no effective vaccine or specific therapy available against hRSV, with the noticeable exception of palivizumab, a humanized monoclonal antibody directed against the F glycoprotein of the virus. Palivizumab is administered by intramuscular injection to high-risk infants during the epidemic season as a prophylactic treatment, but its use is limited by a low benefit-cost ratio (Collins and Melero, 2011). Several compounds inhibiting de novo pyrimidine biosynthesis were recently characterized as broad-spectrum antivirals (Bonavia et al., 2011; Hoffmann et al., 2011; Lucas-Hourani et al., 2013, 2015; Marschall et al., 2013; Munier-Lehmann et al., 2015; Ortiz-Riano et al., 2014; Smee et al., 2012; Wang et al., 2011). Most of these molecules target the fourth enzyme of this metabolic pathway,
C. Grandin et al. / Antiviral Research 125 (2016) 58e62
called DHODH (dihydroorotate dehydrogenase), and were shown to inhibit in vitro a large panel of RNA and DNA viruses, including hRSV (Bonavia et al., 2011). Pyrimidine nucleosides are essential precursors for nucleic acid synthesis, they act as intermediates in multiple metabolic pathways, and depleting cells of pyrimidine nucleosides can boost the innate immune response to viruses (Munier-Lehmann et al., 2013). Altogether, this explains the antiviral activity of pyrimidine biosynthesis inhibitors. Despite promising results in vitro, the antiviral efficacy of these antimetabolites in vivo remains controversial. Various pyrimidine
59
biosynthesis inhibitors failed to reduce the replication of dengue virus, influenza virus or hRSV in experimentally-infected rodents (Bonavia et al., 2011; Smee et al., 2012; Wang et al., 2011), although one compound efficiently inhibited cytomegalovirus replication in mice (Marschall et al., 2013). Controversial results were also obtained with leflunomide, a drug that inhibits both tyrosine kinases and de novo pyrimidine biosynthesis, in rodent models of hRSV infection (Cox Dunn et al., 2011; Davis et al., 2006). Thus, additional experimentations are needed to determine if pyrimidine biosynthesis inhibitors are effective in vivo against virus infections, and
Fig. 1. In vitro inhibition of hRSV growth by IPPA17-A04 and GAC50. (A) Chemical structures of GAC50 and IPPA17-A04. (B and C) hRSV growth was determined in Hep2 cells infected with hRSV (strain Long; MOI ¼ 0.1), and cultured with DMSO alone, or increasing concentrations of IPPA17-A04 (B) or GAC50 (C). Viral RNA copies were determined after 72 h of culture by RT-qPCR as previously described (Grandin et al., 2015). Data show means ± SD of one representative experiment performed in triplicate. ** indicates p-values < 0.01 as determined by standard t-test (D) Viral protein expression in hRSV-infected LLC-MK2 cells (MOI ¼ 0.1) when treated with IPPA17-A04, GAC50 or left untreated (DMSO alone). hRSV-F protein expression was determined by immunostaining using a mouse monoclonal antibody (MAB8599; clone 131-2A).
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hRSV in particular. We recently set up a model of hRSV infection in cynomolgus macaques where the virus is administered by the intranasal route and replicates for less than 15 days in the upper respiratory tract without inducing any pathology (Grandin et al., 2015). We took advantage of this mild-infection model to evaluate the antiviral
potential of IPPA17-A04 and GAC50 (Fig. 1A), two optimized pyrimidine biosynthesis inhibitors developed in our laboratory (Lucas-Hourani et al., 2013, 2015; Munier-Lehmann et al., 2015). This model was selected because GAC50 is only active on primate cells (data not shown). Furthermore, the antiviral activity of pyrimidine biosynthesis inhibitors has never been evaluated in
Fig. 2. Evaluation of palivizumab, IPPA17-A04 and GAC50 antiviral activity in the hRSV-infection macaque model. (A) Timeline of infection, samples collection and treatments. Oneyear old animals were infected intranasally with 5.10þ4 TCID50 of hRSV, and treated with palivizumab, IPPA17-A04 or GAC50 from day 2e9 post-infection. Nasal swabs were collected on all animals at indicated time-points. (BeE) Viral RNA copies detected in nasal swab specimens from control animals (B), or animals treated intranasally with palivizumab (C), IPPA17-A04 (D), or GAC50 (E). hRSV RNA copies in nasal swab specimens were quantified by RT-qPCR, and normalized relative to cell count in each sample as previously described (Grandin et al., 2015). Results correspond to the average of viral RNA copies (log values) from left and right nostrils of each animal. Control animals in (B) were left untreated (55 and 56) or treated with a PBS þ 10% Kolliphor solution (57, 58, and 59), which is used as a carrier for IPPA17-A04 and GAC50. (F) Means of viral RNA copies (log values) for each group of animals. The control curve corresponds to the average of viral RNA copies from all control animals (55e59). (G) Nasal swab specimens collected at day 12 from animals left untreated (55e59) or treated with IPPA17-A04 (60e64) or GAC50 (65e69) were clarified by centrifugation, and then tested at a 1/8 dilution for their antiviral activity in a luciferase-based virus growth assay (Lucas-Hourani et al., 2014). This assay relies on HEK-293T cell cultures infected by a recombinant strain of measles virus expressing luciferase, and is highly sensitive to pyrimidine biosynthesis inhibitors. Culture medium was supplemented or not with uridine at 20 mg/ml.
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primates before and in this model, it was possible to deliver the drug topically by intranasal sprays at the site of virus replication. Prior to in vivo experimentations, compounds were assessed in vitro for hRSV growth inhibition, cytoxicity and metabolic stability. First, IPPA17-A04 and GAC50 were shown to block hRSV replication in human Hep2 cells (Fig. 1B and C), and IC50s were estimated to 5 nM and 25 mM, respectively. The inhibition of hRSV growth was confirmed in the macaque cell line LLC-MK2 by anti-F immunostaining (Fig. 1D). In parallel, the half maximal cytotoxic concentration (CC50) of the compounds was determined using a commercial cell viability assay (CellTiter-Glo reagent; Promega). CC50s of IPPA17-A04 and GAC50 were >500 and 250 mM, respectively, both in Hep2 and LLC-MK2 cells. Metabolic stabilities of the compounds were determined on human liver microsomes (TechMedILL, Illkirch). The half-life of IPPA17-A04 was good (42 min), but GAC50 was rapidly metabolized (11 min). Because IPPA17-A04 showed a higher activity and better metabolic stability than GAC50, we documented its general toxicity and pharmacokinetic in macaques. IPPA17-A04 was injected intravenously to three animals during 6 days at 20 mg/kg/day, and plasmatic concentrations were measured by mass spectrometry at seven timepoints after the first and the last injection (Oroxcell, Romainville). The mean apparent terminal half-life of IPPA17-A04 in plasma was 1.2 ± 0.13 h after the first injection, but increased to 10.1 ± 3.11 h for the last injection, suggesting an accumulation of the molecule in tissues after multiple daily injections. Macroscopic and histopathological studies of the animal tissues after the 6th injection showed no sign of toxicity (Novotec, Lyon). Since IPPA17-A04, our most active and stable molecule, was not toxic for macaques, the two compounds were evaluated for their antiviral activity in vivo. Four groups of five animals were infected with hRSV as previously described (Grandin et al., 2015), and treated from day 2e9 post-infection with IPPA17-04, GAC50, palivizumab, or were left untreated (Fig. 2A). IPPA17-A04 and GAC50 were formulated in PBS þ10% Kolliphor EL at 1 mg/ml, below their limits of solubility (respectively 1.7 and 1.47 mg/ml; TechMedILL), whereas palizivumab was resuspended in PBS at 2.5 mg/ml. Treatments were administered topically in the upper respiratory tract with an intranasal sprayer (MicroSprayer Aerosolizer e Model IA-1B; PennCentury) to target the virus replication site. Animals received once a day in each nostril 1.25 mg of palivizumab, or 0.5 mg of IPPA17-A04 or GAC50. This approximately corresponds to 0.2 mg/ kg/day, a regimen comparable to intranasal doses previously administered in human when evaluating influenza virus or rhinovirus inhibitors with in vitro antiviral activities in the nM to mM range (Calfee et al., 1999; Hayden et al., 1992, 2003; Matsumoto et al., 1999). Three of the five untreated animals received once a day a matching volume of the PBS þ10% Kolliphor EL solution used to dissolve IPPA17-A04 and GAC50. The two other control animals received no treatment at all. Nasal swabs were collected from both left and right nostrils from day 5 before the infection up to day 29 post-infection. Then, total RNA was extracted from nasal swabs, and the number of hRSV RNA copies for 10þ3 cells determined by RTqPCR (Grandin et al., 2015). This protocol was approved by the Animal Care and Use Committee of VetAgro Sup. In this model, the initial inoculum is detected at day 1 postinfection, and is cleared in about 3 days (Grandin et al., 2015). The rebound in virus load detected at day 4e5 unambiguously demonstrates viral replication. As shown in Fig. 2B, numbers of viral RNA copies were virtually similar at the peak of infection in animals left untreated (55 and 56) or treated with a PBS þ 10% Kolliphor solution (57, 58, and 59). Viral RNA copies in palivizumabtreated animals were found 2.7 to 29 times lower than in controls (Fig. 2C and F), and inhibition was highly significant (two-way ANOVA; p-value ¼ 0.0036). This prooved palivizumab efficacy, and
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also demonstrated that the intranasal spray efficiently delivered the antiviral molecule at the site of virus replication. In contrast, IPPA17-A04 or GAC50 did not show any inhibitory effect on hRSV growth between day 4 and 11 (Fig. 2DeF), and the lower viral load detected at day 3 in compound-treated animals was not statistically significant. In contrast, IPPA17-A04 treatment significantly increased viral loads at the peak of replication from day 5e9 (twoway ANOVA; p-value ¼ 0.0494), suggesting some proviral effects. In conclusion, IPPA17-A04 and GAC50 failed to prevent hRSV growth in macaques. We cannot exclude that tested compounds had no effect because they were quickly metabolized. However, some antiviral activity was still detected with a sensitive virus replication assay (Lucas-Hourani et al., 2014) in nasal swab specimens collected at day 12, i.e. up to 3 days after the last administration (Fig. 2G). This activity was reversed by the addition of uridine, supporting the presence of pyrimidine biosynthesis inhibitors in nasal swab specimens. It could be also that delivered doses were too low or compounds were not absorbed by the epithelial tissue. However, the proviral activity of IPPA17-A04 suggests that this was not the case, at least for this molecule. Altogether, our observations are consistent with the negative results reported in hRSV-infected rodents treated with pyrimidine biosynthesis inhibitors by oral gavage (Bonavia et al., 2011; Davis et al., 2006). How can we explain this setback? Besides de novo pyrimidine biosynthesis, cells can acquire pyrimidine nucleosides by the salvage of nucleobases from catabolic pathways and uptakes from the extracellular pool (Munier-Lehmann et al., 2013). Epithelial cells from the respiratory tract could have their needs in pyrimidine nucleosides fulfilled by these two alternative mechanisms if their metabolic activity is limited. This hypothesis should be supported by in situ quantification of pyrimidine levels in treated macaques, something we have not investigated yet. Finally, results obtained with IPPA17-A04 suggest that in vivo, pyrimidine biosynthesis inhibitors can have proviral effects. One hypothesis is that blocking this metabolic pathway could interfere with cellmediated immunity. Indeed, lymphocytes have important needs in nucleosides when proliferating, and inhibitors of de novo pyrimidine biosynthesis can block their expansion both in vitro and in vivo (Munier-Lehmann et al., 2013). In conclusion, our results challenge the development of pyrimidine biosynthesis inhibitors as a treatment against hRSV infection. Acknowledgments This work was supported by the National Research Agency ^le, the (ANR-2011-RPIB-006-01, Program STING 2.0), Lyonbiopo Institut Carnot Pasteur MI (Program STING), the Institut Pasteur, the et de la Recherche Me dicale, and the Institut National de la Sante Centre National de la Recherche Scientifique. CG was supported by a CIFRE fellowship (Conventions Industrielles de Formation par la Recherche, Association Nationale de la Recherche et de la Technologie; 2011/1187). We thank Dr. Jean-François Bureau for his advice on statistical analyses. We thank Dr. Mustapha Si-Tahar and Dr. Nathalie Heuze-Vourc'h for their technical advices on aerosolization technics. References Bonavia, A., Franti, M., Pusateri Keaney, E., Kuhen, K., Seepersaud, M., Radetich, B., Shao, J., Honda, A., Dewhurst, J., Balabanis, K., Monroe, J., Wolff, K., Osborne, C., Lanieri, L., Hoffmaster, K., Amin, J., Markovits, J., Broome, M., Skuba, E., CornellaTaracido, I., Joberty, G., Bouwmeester, T., Hamann, L., Tallarico, J.A., Tommasi, R., Compton, T., Bushell, S.M., 2011. Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV). Proc. Natl. Acad. Sci. U. S. A. 108, 6739e6744. Borchers, A.T., Chang, C., Gershwin, M.E., Gershwin, L.J., 2013. Respiratory syncytial
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