Macromolecular Prodrugs of Ribavirin: StructureâFunction Correlation

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Macromolecular prodrugs of ribavirin: Structure function correlation as inhibitors of influenza infectivity Camilla Frich Riber, Tracey M. Hinton, Paulina Gajda, Kaja #uwa#a, Martin Tolstrup, Cameron Stewart, and Alexander N. Zelikin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00826 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 17, 2016

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Molecular Pharmaceutics

Macromolecular prodrugs of ribavirin: Structure function correlation as inhibitors of influenza infectivity Camilla Frich Riber,1 Tracey M. Hinton,2,* Paulina Gajda,3 Kaja Zuwala,3 Martin Tolstrup,3 Cameron Stewart,2 Alexander N. Zelikin1,4,* 1

Department of Chemistry, Aarhus University, Denmark CSIRO-Health and Biosecurity Business Unit, Australian Animal Health Laboratory, Geelong, Vic 3220 Australia 3 Department of Infectious Diseases, Aarhus University Hospital, Denmark 4 iNano Interdisciplinary Nanoscience Centre, Aarhus University, Aarhus, Denmark Email : [email protected]; [email protected] 2

Abstract The requirement for new antiviral therapeutics is an ever present need. Particularly lacking are broad spectrum antivirals that have low toxicity. We develop such agents based on macromolecular prodrugs whereby both the polymer chain and the drug released from the polymer upon cell entry have antiviral effects. Specifically, macromolecular prodrugs were designed herein based on poly(methacrylic acid) and ribavirin. Structure-function parameter space was analyzed via the synthesis of 10 polymer compositions varied by molar mass and drug content. Antiviral activity was tested in cell culture against both low and high pathogenic strains of influenza. Lead compounds were successfully used to counter infectivity of influenza in chicken embryos. The lead composition with the highest activity against influenza was also active against another respiratory pathogen, respiratory syncytial virus, providing opportunity to potentially treat infection by the two pathogens with one antiviral agent. In contrast, structure-function activity against the herpes simplex virus was drastically different, revealing limitations of the broad spectrum antiviral agents based on macromolecular prodrugs. Keywords Macromolecular prodrugs; Antiviral agents; Drug delivery; Influenza; Respiratory syncytial virus; Herspes simplex virus.

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Introduction Viral pathogens and specifically influenza viruses constitute a tremendous socio-economic burden. 1 Yearly efforts in vaccine development, high levels of mortality particularly among the elderly and the immune-compromised individuals, as well as recurring emergence of highly pathogenic viral mutants make the development of new antiviral agents highly warranted. 2-3 One approach to the design of novel modalities of the antiviral treatment is re-purposing of existing drugs. 4 Further success can be gained using the tools of polymer therapeutics and nanomedicine which may serve to optimize the delivery of therapeutic agents to alleviate toxicity of the treatment. 5-6 One such drug for the treatment of infections by influenza virus is ribavirin. 7-9 This drug has a long history of use in the clinic and has a broad spectrum of activity against several viruses, including influenza virus. However, it is not approved for the treatment of influenza infections, in large part due to a high toxicity of treatment producing hemolytic anemia.8 Over the past few years, we have invested efforts into the optimization of delivery of RBV using macromolecular prodrugs (MP), i.e. polymer-conjugated RBV. 10-16 Polymers were synthesized based on non-ionic N-vinyl pyrrolidone (NVP) and N-(2-hydroxypropyl) methacrylamide (HPMA), as well as anionic acrylic and methacrylic acids (PAA and PMAA, respectively). We have shown that MP exhibit a minimal level of association with red blood cells thus overcoming the origin of the main side effect of the conjugated drug. 10-11 Optimization of the drug conjugation strategy – ester ligation vs disulfide based tether equipped with a self immolative linker – offered significant improvement in terms of the potency of MP. 12 This was verified using an anti-inflammatory model and capitalizing on the ribavirin – nitric oxide connection in macrophages. Polymers based on HPMA and conjugated RBV were effective in suppressing inflammation (in a model for hepatitis) and inhibiting infectivity of HIV. 14 The significance of this dual virus efficacy is underscored by the fact that 25% of the HIV positive patients are reported by the Center for Disease Control, USA to be co-infected with HCV, yet treatments for the co-infection with the two viruses are yet to be designed. Finally, formulations based on the negatively charged PMAA proved to be unique in that the polymer exhibited an inherent antiviral effect and in the overall composition of MP, both the polymer and the drug independently exerted antiviral activity. 16 Indeed, historically, PMAA was identified to have an inherent antiviral effect and this was validated in vivo as far back as the 1960s. Since then, this polymer was shown to be effective as an antiviral agent against Vesicular stomatitis virus (VSV) , Sindbis, Vaccinia,17 and in our own hands against HIV16, 18. Coupled to RBV, an agent with activity against several viral pathogens, PMAA-RBV MP revealed a broad spectrum of antiviral activity 16– a highly sought-after characteristic, specifically in light of increased frequency of emergence of viral pathogens for which there exists no available therapy (e.g. Ebola, Zika). Specifically, PMAA-RBV revealed antiviral effects against influenza virus, human immunodeficiency virus, measles, respiratory syncytial virus, and the Ebola virus. 16 Encouraged by our initial findings, in this work, we systematically investigate the macromolecular parameter space for PMAA-based MP of ribavirin (polymer molar mass and drug content) to elucidate the structure-activity relationship with an ultimate aim to identify the optimal composition of the MP with regards to the potency and efficacy of the antiviral effects. To achieve this goal, a


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library of MP based on PMAA was synthesized with a variation of polymer molar mass, from 6 to 57 kDa, and drug loading, from 0 to 14 mol%. Inhibition of infectivity of the influenza virus was studied for both the low and the high pathogenic virus strains to identify the lead polymer compositions. These were tested in a pre-in vivo chicken embryo model. To investigate if structureactivity relationship is universal for MP as inhibitors of viral infectivity, the synthesized library of polymers was also tested against herpes simplex virus and respiratory syncytial virus. Results presented in this study identify the guidelines for development of successful antiviral agents based on MP of RBV and also highlight the shortcomings of MP as broad spectrum antiviral agents.

Materials and methods Unless mentioned otherwise, all chemicals were purchased from Sigma-Aldrich and used without purification. Organic syntheses: Synthesis of 2-((2-hydroxyethyl)disulfanyl)ethyl methacrylate (1) 2-((2-hydroxyethyl)disulfanyl)ethyl methacrylate was synthesized as described by Kock et al.19 2hydroxyethyldisulphide (11.7 mL, 95.7 mmol, 2 eq.) was dissolved in DCM (250 mL) and flushed with N2. Triethylamine (13.3 mL, 95.7 mmol, 2 eq.) was added followed by methacryloyl chloride (4.7 mL, 47.8 mmol, 1 eq.) which was added dropwise at 0 oC. The reaction was stirred for 1 hour at 0 oC upon which the temperature was increased to room temperature and the mixture stirred further 1.5 hours. The reaction was quenched with ammonium chloride, washed with water and brine. All three aqueous phases were extracted with DCM upon washing. The combined organic phase was dried over anhydrous sodium sulphate and concentrated in vacuo. The crude product was purified by flash column chromatography with EtOAc/Pentane 1:4 to 4:6. The product was dried yielding the pure product as a colorless oil (8.109 g, 36.47 mmol, 76 %). 1H-NMR (400 MHz, CDCl3) δ (ppm) 6.14 (s, 1H), 5.60 (s, 1H), 4.42 (d, J = 6.6 Hz, 2H), 3.89 (m, 2H), 3.02 – 2.92 (m, 2H), 2.88 (m, 2H), 1.95 (s, 3H). Synthesis of 1-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(((tert-butyl-dimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-carboxamide (2). Imidazole (20.9 g, 307 mmol, 3.8 eq) and DMAP (2.14 g, 17.5 mmol, 0.2 eq.) were added to a solution of ribavirin (20.7 g, 68.08 mmol, 1 eq.) in anhydrous DMF (200 mL) under N2 atmosphere. The solution was stirred for 5 minutes upon which a solution of TBDMSCl (78.2 g, 519 mmol, 6.3 eq.) in DMF (140 mL) was added. The reaction mixture was stirred at room temperature for 22 hours. The reaction was monitored by crude 1H-NMR confirming full conversion. The reaction mixture was diluted with DCM,washed 3 times with NH4Cl and once with brine. The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo yielding the crude product which was used in the next step without further purification or characterization.



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Synthesis of 1-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl) tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-carboxamide (3). Compound 2 (68.08 mmol, 1 eq.) was dissolved in MeOH (850 mL) and a solution of HCl (3 mL, 98.3 mmol, 1.5 eq.) in methanol (150 mL) was added and the reaction stirred for 1.5 hours at room temperature. The white precipitate formed during the reaction was filtered and washed with cold methanol. The filtrate was concentrated affording more product which was also filtered and washed with methanol. The combined product was dried in vacuo yielding the pure product as a white solid (29.143 g, 62 mmol, 90 %) 1

H-NMR (400 MHz, CDCl3) δ (ppm) 8.40 (s, 1H), 6.97 (s, 1H), 5.79 (s, 1H), 5.69 (s, 1H), 4.70 (s, 1H), 4.31 (s, 1H), 4.18 (s, 1H), 3.96 (d, J = 12.7 Hz, 1H), 3.72 (d, J = 12.6 Hz, 1H), 0.87 (s, 18H), 0.10 (s, 6H), 0.00 (s, 3H), -0.20 (s, 3H). Synthesis of ((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(3-carbamoyl-1H-1,2,4triazol-1-yl)tetrahydrofuran-2-yl)methyl (4-nitrophenyl) carbonate) (4) Compound 3 (23.7 g, 50.1 mmol, 1 eq.) was suspended in dry THF (200 mL) under N2 atmosphere. Triethylamine (14.5 mL, 104 mmol, 2 eq.) was added and the mixture stirred for 5 min. Paranitrophenyl chloroformate (15.8 g, 78.2 mmol, 1.5 eq.) was added and the reaction stirred at room temperature for 13 hours. The suspension was concentrated and the residue dissolved in EtOAc. The crude mixture was purified by flash column chromatography 7:3 pentane/EtOAc affording the pure product (22.6 g, 35.5 mmol, 71%)

1

H-NMR (400 MHz, CDCl3) δ (ppm) 8.39 (s, 1H), 8.30 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.7 Hz, 2H), 6.95 (s, 1H), 5.80 (s, 1H), 5.61 (s, 1H), 4.71 – 4.53 (m, 2H), 4.39 (m, 2H), 4.12 (m, 1H), 0.91 (m, 18H), 0.27– -0.06 (m, 12H). Synthesis of TBDMS protected RBV(SIL)methacrylate (2-((2-(((((2R,3R,4R,5R)-3,4-bis((tertbutyldimethylsilyl)oxy)-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)tetrahydrofuran -2-yl)methoxy) carbonyl)oxy)ethyl)disulfanyl)ethyl methacrylate) (M1) A solution of 4 (7.97 g, 12.5 mmol, 1 eq.) in DCM (250 mL) was added to a solution of 1 (4.17 g, 18.8 mmol, 1.5 eq.), DIPEA (6.53 mL, 37.5 mmol, 3 eq.) and DMAP (0.305 g, 2.5 mmol, 0.2 eq.) in DCM (600 mL) under N2 atmosphere. The reaction was stirred at room temperature for 24 hours. The reaction mixture was washed twice with NH4Cl, twice with brine, and dried over MgSO4. The crude was purified by flash column chromatography pentane/EtOAc, from 4:1 to 1:1 affording the pure product (8.39 g, 11.6 mmol, 93%) at the same time recovering excess 2-((2-hydroxyethyl)disulfanyl)ethyl methacrylate as the pure reactant. 1

H-NMR (400 MHz, CDCl3) δ (ppm) 8.40 (s, 1H), 7.01 (s, 1H), 6.13 (m, 1H), 5.77 (m, 2H), 5.59 (m, 1H), 4.57 – 4.36 (m, 6H), 4.35 – 4.21 (m, 3H), 2.97 (dt, J = 11.3, 6.7 Hz, 4H), 1.94 (s, 2H), 0.89 (m, 18H), 0.21 – -0.10 (m, 12H).


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Polymerization All PMAA-co-RBV(SIL)MA polymers were synthesized following the same general procedure, described below, with the amounts of reagents used for each polymer synthesis reported in Table 1. Methacrylic acid, TBDMS protected RBV(SIL) methacrylate (M1), initiator of radical polymerization azo-isobutyronitrile (AIBN), and RAFT chain transfer reagent, 4-Cyano-4[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDPA) were dissolved in DMF (0.2 mL). Five rounds of freeze-pump-thaw were performed before the ampule was flame-sealed and the reaction performed at 60 °C for 15 hours. The reaction mixture was subsequently diluted with DMF (1.2 mL), TEA·3HF (0.45 mL) was added and the reaction stirred at room temperature for 20 hours. The solution was precipitated into DCM with 5% methanol yielding the pure product. 1

H-NMR (400 MHz, DMSO) δ (ppm) 8.82 (s), 7.87 (s), 7.66 (s), 5.91 (m), 4.55 – 3.99 (m), 2.08 – 0.34 (m, 5H). Table 1. Compositions of reaction mixtures prepared for the synthesis of PMAA-RBV prodrugs, resulting conversions and recovered mass for the prepared polymers. n(MAA) (mmol) PMAA-R1 PMAA-R2 PMAA-R3 PMAA-R4 PMAA-R5 PMAA-R6 PMAA-R7 PMAA-R8 PMAA-R9

1.72 1.72 1.72 1.72 1.01 1.00 1.01 1.01 1.01

n(AIBN) n(CDPA) (mmol) (mmol) 0.00801 0.00809 0.004 0.004 0.00237 0.00114 0.00125 0.000791 0.000333

0.0411 0.0414 0.0222 0.0194 0.0116 0.00559 0.00586 0.00385 0.00167

n(RBV-MA) (mmol) 0.495 0.215 0.469 0.221 0.122 0.114 0.116 0.111 0.128

conv. conv. Mass MAA RBV recovered (%) (%) (mg) 78 92 147 81 96 109 65 86 134 76 86 119 89 100 77 80 85 65 83 100 80 82 95 69 24 56 67

Cell culture African green monkey kidney cells (Vero; ATCC No CRL-1587), adenocarcinomic human alveolar basal epithelial cells (A549; ATCC No.CCL-185) and human cervix cancer epithelial cells (HeLa; ATCC No. CCL-2) were grown in Dulbecco’s Modified Essential Medium (DMEM) (Lonza, Basel, Switzerland) containing 10% heat-inactivated fetal calf serum (FCS) and 50 U/ml penicillin and 50 µg/ml streptomycin (cDMEM). Cells were cultured in the atmosphere of 5% CO2, at 37°C. Cell cultures were regularly checked for contamination with mycoplasma using MycoAlert mycoplasma detection kit (Lonza). Inhibition of Influenza A infectivity. Influenza H1N1 A/WSN/33 (WSN; AAHL), Influenza H1N1 A/Puerto Rico/8/1934 (PR8; AAHL) and Influenza H5N1 A/Vietnam/1203/2004 (H5N1) were produced by limiting dilution passage in the allantoic cavity of 10-day old embryonated chicken eggs at 34 °C for 48–72 h. Assays were performed on either Adenocarcinomic human alveolar basal epithelial cells (A549; ATCC No.CCL5 ACS Paragon Plus Environment


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185) or African Green Monkey Kidney cells (Vero; ATCC No CRL-1587). Cells were seeded at 2 x104 cells per well in 96-well tissue culture plates and grown overnight at 37 ºC with 5 % CO2. Polymers at required concentrations were added 1-2 h before the virus in triplicate. For low MOI experiments infection continued for 48 h, for high MOI experiments infection lasted for 24 h. Supernatant was collected and stored at -80°C, the plates were fixed with 4% paraformaldehyde (PFA) in PBS for 1 h. Cells were permeabilized with 0.1 % Triton X-100 (Sigma, St. Louis, MO) in PBS for 30 min and immunofluorecence staining was performed, using following fluorescent antibodies and stains:primary mouse anti-Influenza H1 NP antibody (AbD Serotec Kidlington, UK), secondary goat anti-mouse AF488 antibody (Life Technologies, Carlsbad, CA, USA) and 4',6diamidino-2-phenylindole (DAPI, Invitrogen, USA) The 96-well plates were imaged by the Cell Insight system (Thermofisher Scientific, USA) at a magnification of 10 x. 49 fields/well were imaged representing the entire well. The percentage of infected cells from 75,000 nuclei counted was quantified using the Target Activation bioapplication of the Cellomics Scan software (iDev workflow) and was determined by dividing the number of antigen-positive cells by the total cell number, multiplied by 100. In vivo influenza virus silencing. Polymers at 2 mg/L in 50 µL PBS were injected into the allantoic cavity of 10-day old embryonated chicken eggs based on 6ml allantoic fluid. H1N1 Influenza PR8 virus was diluted in 50 µL PBS to 500 pfu/egg and immediately injected into the allantoic cavity of the treated embryonated chicken eggs. The eggs were incubated at 37 °C for 48 h. Embryo were euthanised on ice O/N and allantoic fluid was harvested to measure virus titre. TCID50 assays were performed as described in 20. Briefly, tissue culture supernatants or allantoic fluid were assayed for virus infectivity on Madin-Darby canine kidney cells (MDCK: ATCC No. CCL-34) by endpoint dilution for cytopathic effect with a 10-fold dilution series after 72 h. The infectious titre was calculated by the method of Reed and Muench 21. Titres are expressed as log10 TCID50/ml ± SEM.

Inhibition of Respiratory Syncytial Virus infectivity. Respiratory Syncytial virus (RSV, ATCC VR1540) was produced and assays were performed on adenocarcinomic human alveolar basal epithelial cells (A549; ATCC No.CCL-185). A549 cells were seeded at 2 x104 cells per well in 96-well tissue culture plates and grown overnight at 37 ºC with 5 % CO2. Assays were performed as described above for detection of influenza A virus infectivity. Immunofluorecence staining was performed and analysed as above for detection of influenza A virus infectivity with one exception: the primary antibody used was rabbit polyclonal antibody produced at AAHL against the P protein (AAHL, Australia).

Inhibition of herpes simplex virus type 2 infectivity Vero cells were seeded on 24-well plate at the density of 2·105 cells per well and cultured overnight. After overnight culture the media was replaced with fresh one. Virus was incubated with the drugs


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at indicated concentrations for 30 min at the bench and then added to the cells. 24 hours later a standard plaque assay was performed. Briefly, media was removed and the cells were fixed in 4% PFA solution for 10 min. Subsequently the cells were washed twice with PBS. 0.5% solution of crystal violet in PBS containing 10 % ethanol was used to stain the cells for 10 min. The stain was washed away with PBS. Plaques were enumerated under the microscope. The number of plaques was compared to the control sample only infected with HSV-2 and presented as percentage of the control.

Cell Viability Assays. Toxicity was measured using the Alamar Blue reagent (Invitrogen, Carlsbad, CA) or PrestoBlue reagent (Invitrogen) according to manufacturer’s instructions. In case of Alamar Blue reagent the media was removed and replaced with 100 µL of standard media containing 10% Alamar Blue reagent, cells were then incubated for 4 h at 37 ºC with 5 % CO2. The assay was read on an EL808 Absorbance microplate reader (BIOTEK, Winooski, VT) at 540 nm and 620 nm. Cell viability was determined by subtracting the 620 nm measurement from the 540 nm measurement.. In case of PrestoBlue reagent, the media was removed and replaced with 100 µL of cDMEM containing 10% of PrestoBlue reagent. The cells were incubated for 30 min at 37°C. The fluorescence (excitation: 560 nm, emission: 590 nm) as a measure of viability was quantified using FLUOstar Omega plate reader (BMG Labtech, Ortenberg, Germany). Results are presented as a percentage of untreated cells and the presented data are representative of three separate experiments in triplicate. Statistics. The difference between two groups was statistically analysed by one way repeated measures ANOVA, parametric, with Dunnet post analysis against virus only cells. *** p< 0.001, **p

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