108 results on '"Babusis, Darius"'
Search Results
2. Pharmacokinetic, Pharmacodynamic, and Drug-Interaction Profile of Remdesivir, a SARS-CoV-2 Replication Inhibitor
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Humeniuk, Rita, Mathias, Anita, Kirby, Brian J., Lutz, Justin D., Cao, Huyen, Osinusi, Anu, Babusis, Darius, Porter, Danielle, Wei, Xuelian, Ling, John, Reddy, Y. Sunila, and German, Polina
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- 2021
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3. The oral nucleoside prodrug GS-5245 is efficacious against SARS-CoV-2 and other endemic, epidemic, and enzootic coronaviruses.
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Martinez, David R., Moreira, Fernando R., Catanzaro, Nicholas J., Diefenbacher, Meghan V., Zweigart, Mark R., Gully, Kendra L., De la Cruz, Gabriela, Brown, Ariane J., Adams, Lily E., Yount, Boyd, Baric, Thomas J., Mallory, Michael L., Conrad, Helen, May, Samantha R., Dong, Stephanie, Scobey, D. Trevor, Nguyen, Cameron, Montgomery, Stephanie A., Perry, Jason K., and Babusis, Darius
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SARS-CoV-2 ,PRODRUGS ,CORONAVIRUS diseases ,MERS coronavirus ,SARS-CoV-2 Omicron variant ,CORONAVIRUSES ,RNA replicase - Abstract
Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV–related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (M
pro ) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease. Editor's summary: Currently approved antivirals for SARS-CoV-2 target one of two viral proteins. Remdesivir and molnupiravir target the RNA-dependent RNA polymerase (RdRp), whereas nirmatrelvir (the antiviral agent of Paxlovid) targets the main protease (Mpro ). Although these drugs are effective, there continues to be room for improvement, especially for drugs targeting the RdRp. Here, Martinez et al. evaluated the efficacy of an orally available small-molecule targeting the RdRp of SARS-CoV-2 and other coronaviruses called GS-5245 or obeldesivir. The authors found that GS-5245 could reduce disease severity in mice infected with one of several different coronaviruses, including SARS-CoV-2, SARS-CoV, and MERS-CoV. Moreover, combining GS-5245 with nirmatrelvir further improved outcomes in mice infected with SARS-CoV-2. Together, these data support further development of GS-5245/obeldesivir as a broader anti-coronaviral drug. —Courtney Malo [ABSTRACT FROM AUTHOR]- Published
- 2024
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4. Oral prodrug of remdesivir parent GS-441524 is efficacious against SARS-CoV-2 in ferrets
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Cox, Robert M., Wolf, Josef D., Lieber, Carolin M., Sourimant, Julien, Lin, Michelle J., Babusis, Darius, DuPont, Venice, Chan, Julie, Barrett, Kim T., Lye, Diane, Kalla, Rao, Chun, Kwon, Mackman, Richard L., Ye, Chengjin, Cihlar, Tomas, Martinez-Sobrido, Luis, Greninger, Alexander L., Bilello, John P., and Plemper, Richard K.
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- 2021
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5. Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV
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Sheahan, Timothy P., Sims, Amy C., Leist, Sarah R., Schäfer, Alexandra, Won, John, Brown, Ariane J., Montgomery, Stephanie A., Hogg, Alison, Babusis, Darius, Clarke, Michael O., Spahn, Jamie E., Bauer, Laura, Sellers, Scott, Porter, Danielle, Feng, Joy Y., Cihlar, Tomas, Jordan, Robert, Denison, Mark R., and Baric, Ralph S.
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- 2020
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6. Chemoprophylaxis With Oral Emtricitabine and Tenofovir Alafenamide Combination Protects Macaques From Rectal Simian/Human Immunodeficiency Virus Infection
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Massud, Ivana, Mitchell, James, Babusis, Darius, Deyounks, Frank, Ray, Adrian S., Rooney, James F., Heneine, Walid, Miller, Michael D., and García-Lerma, J. Gerardo
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- 2016
7. Discovery of GS-5245 (Obeldesivir), an Oral Prodrug of Nucleoside GS-441524 That Exhibits Antiviral Efficacy in SARS-CoV-2-Infected African Green Monkeys.
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Mackman, Richard L., Kalla, Rao V., Babusis, Darius, Pitts, Jared, Barrett, Kimberly T., Chun, Kwon, Du Pont, Venice, Rodriguez, Lauren, Moshiri, Jasmine, Xu, Yili, Lee, Michael, Lee, Gary, Bleier, Blake, Nguyen, Anh-Quan, O'Keefe, B. Michael, Ambrosi, Andrea, Cook, Meredith, Yu, Joy, Dempah, Kassibla Elodie, and Bunyan, Elaine
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- 2023
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8. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys
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Warren, Travis K., Jordan, Robert, Lo, Michael K., Ray, Adrian S., Mackman, Richard L., Soloveva, Veronica, Siegel, Dustin, Perron, Michel, Bannister, Roy, Hui, Hon C., Larson, Nate, Strickley, Robert, Wells, Jay, Stuthman, Kelly S., Van Tongeren, Sean A., Garza, Nicole L., Donnelly, Ginger, Shurtleff, Amy C., Retterer, Cary J., Gharaibeh, Dima, Zamani, Rouzbeh, Kenny, Tara, Eaton, Brett P., Grimes, Elizabeth, Welch, Lisa S., Gomba, Laura, Wilhelmsen, Catherine L., Nichols, Donald K., Nuss, Jonathan E., Nagle, Elyse R., Kugelman, Jeffrey R., Palacios, Gustavo, Doerffler, Edward, Neville, Sean, Carra, Ernest, Clarke, Michael O., Zhang, Lijun, Lew, Willard, Ross, Bruce, Wang, Queenie, Chun, Kwon, Wolfe, Lydia, Babusis, Darius, Park, Yeojin, Stray, Kirsten M., Trancheva, Iva, Feng, Joy Y., Barauskas, Ona, Xu, Yili, Wong, Pamela, Braun, Molly R., Flint, Mike, McMullan, Laura K., Chen, Shan-Shan, Fearns, Rachel, Swaminathan, Swami, Mayers, Douglas L., Spiropoulou, Christina F., Lee, William A., Nichol, Stuart T., Cihlar, Tomas, and Bavari, Sina
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- 2016
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9. The Nucleoside/Nucleotide Analogs Tenofovir and Emtricitabine Are Inactive against SARS-CoV-2.
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Feng, Joy Y., Du Pont, Venice, Babusis, Darius, Gordon, Calvin J., Tchesnokov, Egor P., Perry, Jason K., Duong, Vincent, Vijjapurapu, Arya, Zhao, Xiaofeng, Chan, Julie, Cohen, Cal, Juneja, Kavita, Cihlar, Tomas, Götte, Matthias, and Bilello, John P.
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ANTIVIRAL agents ,EMTRICITABINE ,SARS-CoV-2 ,EMTRICITABINE-tenofovir ,REVERSE transcriptase ,REVERSE transcriptase inhibitors - Abstract
The urgent response to the COVID-19 pandemic required accelerated evaluation of many approved drugs as potential antiviral agents against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using cell-based, biochemical, and modeling approaches, we studied the approved HIV-1 nucleoside/tide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) and emtricitabine (FTC), as well as prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxilfumarate (TDF) for their antiviral effect against SARS-CoV-2. A comprehensive set of in vitro data indicates that TFV, TAF, TDF, and FTC are inactive against SARS-CoV-2. None of the NRTIs showed antiviral activity in SARS-CoV-2 infected A549-hACE2 cells or in primary normal human lung bronchial epithelial (NHBE) cells at concentrations up to 50 µM TAF, TDF, FTC, or 500 µM TFV. These results are corroborated by the low incorporation efficiency of respective NTP analogs by the SARS-CoV-2 RNA-dependent-RNA polymerase (RdRp), and lack of the RdRp inhibition. Structural modeling further demonstrated poor fitting of these NRTI active metabolites at the SARS-CoV-2 RdRp active site. Our data indicate that the HIV-1 NRTIs are unlikely direct-antivirals against SARS-CoV-2, and clinicians and researchers should exercise caution when exploring ideas of using these and other NRTIs to treat or prevent COVID-19. [ABSTRACT FROM AUTHOR]
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- 2022
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10. Therapeutic treatment with an oral prodrug of the remdesivir parental nucleoside is protective against SARS-CoV-2 pathogenesis in mice.
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Schäfer, Alexandra, Martinez, David R., Won, John J., Meganck, Rita M., Moreira, Fernando R., Brown, Ariane J., Gully, Kendra L., Zweigart, Mark R., Conrad, William S., May, Samantha R., Dong, Stephanie, Kalla, Rao, Chun, Kwon, Du Pont, Venice, Babusis, Darius, Tang, Jennifer, Murakami, Eisuke, Subramanian, Raju, Barrett, Kimberly T., and Bleier, Blake J.
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COVID-19 ,SARS-CoV-2 ,MONOCLONAL antibodies ,RNA polymerases ,MIDDLE East respiratory syndrome ,ORAL drug administration ,REMDESIVIR ,RNA replicase - Abstract
The coronavirus disease 2019 (COVID-19) pandemic remains uncontrolled despite the rapid rollout of safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. In addition, the emergence of SARS-CoV-2 variants of concern, with their potential to escape neutralization by therapeutic monoclonal antibodies, emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parent nucleoside of remdesivir, which targets the highly conserved virus RNA-dependent RNA polymerase. GS-621763 exhibited antiviral activity against SARS-CoV-2 in lung cell lines and two different human primary lung cell culture systems. GS-621763 was also potently antiviral against a genetically unrelated emerging coronavirus, Middle East respiratory syndrome CoV (MERS-CoV). The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 administration reduced viral load and lung pathology; treatment also improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral that has recently received EUA approval, proved both drugs to be similarly efficacious in mice. These data support the exploration of GS-441524 oral prodrugs for the treatment of COVID-19. A new route for remdesivir: Remdesivir, an antiviral drug that targets the RNA-dependent RNA polymerase of SARS-CoV-2, is an important therapeutic option for individuals with COVID-19. However, the need to administer remdesivir through the intravenous route has limited its use. Here, Schäfer and colleagues tested the efficacy of an oral prodrug of the remdesivir parental nucleoside, GS-621763, against SARS-CoV-2, showing that it was protective in vitro and in vivo. GS-621763 also prevented replication of another coronavirus, MERS-CoV, in vitro. GS-621763 was as efficacious as molnupiravir, another oral antiviral treatment, at reducing disease in mice infected with SARS-CoV-2, warranting further study of GS-621763. [ABSTRACT FROM AUTHOR]
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- 2022
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11. Inhaled remdesivir reduces viral burden in a nonhuman primate model of SARS-CoV-2 infection.
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Vermillion, Meghan S., Murakami, Eisuke, Ma, Bin, Pitts, Jared, Tomkinson, Adrian, Rautiola, Davin, Babusis, Darius, Irshad, Hammad, Seigel, Dustin, Kim, Cynthia, Zhao, Xiaofeng, Niu, Congrong, Yang, Jesse, Gigliotti, Andrew, Kadrichu, Nani, Bilello, John P., Ellis, Scott, Bannister, Roy, Subramanian, Raju, and Smith, Bill
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COVID-19 ,REMDESIVIR ,SARS-CoV-2 ,CERCOPITHECUS aethiops ,ANTIVIRAL agents ,FLUTICASONE ,COVID-19 treatment ,INTRAVENOUS therapy - Abstract
Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the U.S. FDA approved intravenous RDV as the first treatment for hospitalized COVID-19 patients. Furthermore, RDV has been approved or authorized for emergency use in more than 50 countries. To make RDV more convenient for nonhospitalized patients earlier in disease, alternative routes of administration are being evaluated. Here, we investigated the pharmacokinetics and efficacy of RDV administered by head dome inhalation in African green monkeys (AGM). Relative to an intravenous administration of RDV at 10 mg/kg, an about 20-fold lower dose administered by inhalation produced comparable concentrations of the pharmacologically active triphosphate in lower respiratory tract tissues. Distribution of the active triphosphate into the upper respiratory tract was also observed after inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with intravenous RDV dosing. An efficacy study with repeated dosing of inhaled RDV in an AGM model of SARS-CoV-2 infection demonstrated reductions in viral replication in bronchoalveolar lavage fluid and respiratory tract tissues compared with placebo. Efficacy was observed with inhaled RDV administered once daily at a pulmonary deposited dose of 0.35 mg/kg beginning about 8 hours after infection. Moreover, the efficacy of inhaled RDV was similar to that of intravenous RDV administered once at 10 mg/kg followed by 5 mg/kg daily in the same study. Together, these findings support further clinical development of inhalation RDV. An inhaled antiviral: The antiviral drug, remdesivir, has demonstrated clinical benefit in patients diagnosed with COVID-19. However, the requirement of intravenous delivery complicates administration of remdesivir. To address this, Vermillion et al. tested the efficacy of remdesivir when administered via inhalation. The authors treated African green monkeys infected with SARS-CoV-2 with remdesivir using a head dome, showing that it reduced viral load as compared with placebo. Further, inhaled and intravenous remdesivir showed similar efficacy when administered at the same time point. Together, these results support further clinical investigation of inhaled remdesivir for COVID-19. [ABSTRACT FROM AUTHOR]
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- 2022
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12. Oral administration of obeldesivir protects nonhuman primates against Sudan ebolavirus.
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Cross, Robert W., Woolsey, Courtney, Chu, Victor C., Babusis, Darius, Bannister, Roy, Vermillion, Meghan S., Geleziunas, Romas, Barrett, Kimberly T., Bunyan, Elaine, Nguyen, Anh-Quan, Cihlar, Tomas, Porter, anielle P., Prasad, Abhishek N., Deer, Daniel J., Borisevich, Viktoriya, Agans, Krystle N., Martinez, Jasmine, Harrison, Mack B., Dobias, Natalie S., and Fenton, Karla A.
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- 2024
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13. Two-dose emtricitabine/tenofovir alafenamide plus bictegravir prophylaxis protects macaques against SHIV infection.
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Bekerman, Elena, Cox, Stephanie, Babusis, Darius, Campigotto, Federico, Das, Moupali, Barouch, Dan H, Cihlar, Tomas, and Callebaut, Christian
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Objectives: Current prophylaxis options for people at risk for HIV infection include two US FDA-approved daily pre-exposure prophylaxis (PrEP) regimens and guidelines for a 2-1-1 event-driven course specifically for men who have sex with men. Despite this, PrEP use rates remain suboptimal, and additional PrEP options may help to improve uptake among diverse populations. Here, we evaluated protective efficacy of two-dose PrEP and two-dose postexposure prophylaxis (PEP) schedules with emtricitabine (FTC)/tenofovir alafenamide (TAF) with or without bictegravir (BIC) in an SHIV macaque model.Methods: Macaques received one oral dose of 200 mg emtricitabine, 25 mg tenofovir alafenamide and 25-100 mg of bictegravir to establish pharmacokinetic profiles of each drug either in the plasma or the peripheral blood mononuclear cells. Protective efficacy of multiple two-dose PrEP and PEP schedules with FTC/TAF with or without bictegravir was then assessed in two repeat low-dose rectal SHIV challenge studies.Results: The data revealed over 95% per-exposure risk reduction with FTC/TAF PrEP initiated 2 h before the exposure, but a loss of significant protection with treatment initiation postexposure. In contrast, FTC/TAF plus BIC offered complete protection as PrEP and greater than 80% per-exposure risk reduction with treatment initiation up to 24 h postexposure.Conclusions: Together, these results demonstrate that two-dose schedules can protect macaques against SHIV acquisition and highlight the protective advantage of adding the integrase inhibitor bictegravir to the reverse transcriptase inhibitors emtricitabine and tenofovir alafenamide as part of event-driven prophylaxis. [ABSTRACT FROM AUTHOR]- Published
- 2021
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14. Discovery of a 2′-fluoro-2′-C-methyl C-nucleotide HCV polymerase inhibitor and a phosphoramidate prodrug with favorable properties
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Kirschberg, Thorsten A., Metobo, Sammy, Clarke, Michael O., Aktoudianakis, Vangelis, Babusis, Darius, Barauskas, Ona, Birkus, Gabriel, Butler, Thomas, Byun, Daniel, Chin, Gregory, Doerffler, Edward, Edwards, Thomas E., Fenaux, Martijn, Lee, Rick, Lew, Willard, Mish, Michael R., Murakami, Eisuke, Park, Yeojin, Squires, Neil H., Tirunagari, Neeraj, Wang, Ting, Whitcomb, Mark, Xu, Jie, Yang, Huiling, Ye, Hong, Zhang, Lijun, Appleby, Todd C., Feng, Joy Y., Ray, Adrian S., Cho, Aesop, and Kim, Choung U.
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- 2017
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15. Efficacy of Oral Tenofovir Alafenamide/Emtricitabine Combination or Single-Agent Tenofovir Alafenamide Against Vaginal Simian Human Immunodeficiency Virus Infection in Macaques.
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Massud, Ivana, Cong, Mian-Er, Ruone, Susan, Holder, Angela, Dinh, Chuong, Nishiura, Kenji, Khalil, George, Pan, Yi, Lipscomb, Jonathan, Johnson, Ryan, Deyounks, Frank, Rooney, James F, Babusis, Darius, Park, Yeojin, McCallister, Scott, Callebaut, Christian, Heneine, Walid, and García-Lerma, J Gerardo
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HIV infections ,SIMIAN immunodeficiency virus ,MACAQUES ,TENOFOVIR ,HIV - Abstract
Background: Tenofovir alafenamide (TAF)-based regimens are being evaluated for pre-exposure prophylaxis (PrEP). We used a macaque model of repeated exposures to simian human immunodeficiency virus (SHIV) to investigate whether TAF alone or the combination of TAF and emtricitabine (FTC) can prevent vaginal infection.Methods: Pigtail macaques were exposed vaginally to SHIV162p3 once a week for up to 15 weeks. Animals received clinical doses of FTC/TAF (n = 6) or TAF (n = 9) orally 24 hours before and 2 hours after each weekly virus exposure. Infection was compared with 21 untreated controls.Results: Five of the 6 animals in the FTC/TAF and 4 of the 9 animals in the TAF alone group were protected against infection (P = .001 and P = .049, respectively). The calculated efficacy of FTC/TAF and TAF was 91% (95% confidence interval [CI], 34.9%-98.8%) and 57.8% (95% CI, -8.7% to 83.6%), respectively. Infection in FTC/TAF but not TAF-treated macaques was delayed relative to controls (P = .005 and P = .114). Median tenofovir diphosphate (TFV-DP) levels in peripheral blood mononuclear cells (PBMCs) were similar among infected and uninfected macaques receiving TAF PrEP (351 and 143 fmols/106 cells, respectively; P = .921).Conclusions: Emtricitabine/TAF provided a level of protection against vaginal challenge similar to FTC/TFV disoproxil fumarate combination in the macaque model. Our results support the clinical evaluation of FTC/TAF for PrEP in women. [ABSTRACT FROM AUTHOR]- Published
- 2019
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16. Synthesis and characterization of 1′-C-cyano-2′-fluoro-2′-C-methyl pyrimidine nucleosides as HCV polymerase inhibitors
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Kirschberg, Thorsten A., Mish, Michael R., Zhang, Lijun, Squires, Neil H., Wang, Ke-Yu, Cho, Aesop, Feng, Joy Y., Fenaux, Martijn, Babusis, Darius, Park, Yeojin, Ray, Adrian S., and Kim, Choung U.
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- 2015
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17. Preparation and biological evaluation of 1′-cyano-2′-C-methyl pyrimidine nucleosides as HCV NS5B polymerase inhibitors
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Mish, Michael R., Cho, Aesop, Kirschberg, Thorsten, Xu, Jie, Sebastian Zonte, C., Fenaux, Martijn, Park, Yeojin, Babusis, Darius, Feng, Joy Y., Ray, Adrian S., and Kim, Choung U.
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- 2014
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18. Evaluation of 2′-α-fluorine modified nucleoside phosphonates as potential inhibitors of HCV polymerase
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Parrish, Jay P., Lee, Sharon K., Boojamra, Constantine G., Hui, Hon, Babusis, Darius, Brown, Brandon, Shih, I-hung, Feng, Joy Y., Ray, Adrian S., and Mackman, Richard L.
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- 2013
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19. Isosteric analogs of lenalidomide and pomalidomide: Synthesis and biological activity
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Ruchelman, Alexander L., Man, Hon-Wah, Zhang, Weihong, Chen, Roger, Capone, Lori, Kang, Jian, Parton, Anastasia, Corral, Laura, Schafer, Peter H., Babusis, Darius, Moghaddam, Mehran F., Tang, Yang, Shirley, Michael A., and Muller, George W.
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- 2013
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20. Synthesis and characterization of 2′-C-Me branched C-nucleosides as HCV polymerase inhibitors
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Cho, Aesop, Zhang, Lijun, Xu, Jie, Babusis, Darius, Butler, Thomas, Lee, Rick, Saunders, Oliver L., Wang, Ting, Parrish, Jay, Perry, Jason, Feng, Joy Y., Ray, Adrian S., and Kim, Choung U.
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- 2012
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21. Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.
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Sheahan, Timothy P., Sims, Amy C., Graham, Rachel L., Menachery, Vineet D., Gralinski, Lisa E., Case, James B., Leist, Sarah R., Pyrc, Krzysztof, Feng, Joy Y., Trantcheva, Iva, Bannister, Roy, Park, Yeojin, Babusis, Darius, Clarke, Michael O., Mackman, Richard L., Spahn, Jamie E., Palmiotti, Christopher A., Siegel, Dustin, Ray, Adrian S., and Cihlar, Tomas
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CORONAVIRUSES ,RESPIRATORY infections ,DISEASE prevalence ,PREVENTIVE medicine ,PUBLIC health administration ,DISEASE risk factors - Abstract
The article explores on the prevalence of genetically diverse coronavirus in a variety of mammals such as birds and humans. It highlights the availability of effective treatment to prevent the growth of coronavirus. It also cites the antiviral activity and cytotoxicity of the treatment for coronavirus.
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- 2017
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22. Viability of primary osteoblasts after treatment with tenofovir alafenamide: Lack of cytotoxicity at clinically relevant drug concentrations.
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Callebaut, Christian, Liu, Yang, Babusis, Darius, Ray, Adrian, Miller, Michael, and Kitrinos, Kathryn
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OSTEOBLASTS ,TENOFOVIR ,CELL-mediated cytotoxicity ,PRODRUGS ,REVERSE transcriptase inhibitors - Abstract
Tenofovir alafenamide (TAF) is a phosphonoamidate prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV). TAF is approved for the treatment of HIV-1 infection as part of the single-tablet regimen containing elvitegravir, cobicistat, emtricitabine, and TAF. When dosed once-daily, TAF results in approximately 90% lower levels of plasma TFV and a 4-fold increase in intracellular TFV-diphosphate (TFV-DP) in PBMCs compared with the TFV prodrug tenofovir disoproxil fumarate (TDF). Several antiretrovirals, including TDF, have been associated with bone mineral density decreases in patients; the effect of clinically relevant TAF concentrations on primary osteoblast viability was therefore assessed in vitro. Studies in PBMCs determined that a 2-hour TAF exposure at concentrations similar to human plasma C
max achieved intracellular TFV-DP levels comparable to those observed after the maximum recommended human dose of 25 mg TAF. Comparable intracellular TFV-DP levels were achieved in primary osteoblasts with 2-hour TAF exposure daily for 3 days at concentrations similar to those used for PBMCs (100–400 nM). No change in cell viability was observed in either primary osteoblasts or PBMCs. The mean TAF CC50 in primary osteoblasts after 3 days of daily 2-hour pulses was >500 μM, which is >1033 times higher than the TAF maximum recommended human dose plasma Cmax . In summary, primary osteoblasts were not preferentially loaded by TAF compared with PBMCs, with comparable TFV-DP levels achieved in both cell types. Furthermore, there was no impact on osteoblast cell viability at clinically relevant TAF concentrations. [ABSTRACT FROM AUTHOR]- Published
- 2017
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23. Synthesis, anti-HIV activity, and resistance profiles of ribose modified nucleoside phosphonates
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Mackman, Richard L., Boojamra, Constantine G., Prasad, Vidya, Zhang, Lijun, Lin, Kuei-Ying, Petrakovsky, Oleg, Babusis, Darius, Chen, James, Douglas, Janet, Grant, Deborah, Hui, Hon C., Kim, Choung U., Markevitch, David Y., Vela, Jennifer, Ray, Adrian, and Cihlar, Tomas
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- 2007
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24. Intravenous delivery of GS-441524 is efficacious in the African green monkey model of SARS-CoV-2 infection.
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Pitts, Jared, Babusis, Darius, Vermillion, Meghan S., Subramanian, Raju, Barrett, Kim, Lye, Diane, Ma, Bin, Zhao, Xiaofeng, Riola, Nicholas, Xie, Xuping, Kajon, Adriana, Lu, Xianghan, Bannister, Roy, Shi, Pei-Yong, Toteva, Maria, Porter, Danielle P., Smith, Bill J., Cihlar, Tomas, Mackman, Richard, and Bilello, John P.
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COVID-19 , *SARS-CoV-2 , *CERCOPITHECUS aethiops - Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has infected over 260 million people over the past 2 years. Remdesivir (RDV, VEKLURY®) is currently the only antiviral therapy fully approved by the FDA for the treatment of COVID-19. The parent nucleoside of RDV, GS-441524, exhibits antiviral activity against numerous respiratory viruses including SARS-CoV-2, although at reduced in vitro potency compared to RDV in most assays. Here we find in both human alveolar and bronchial primary cells, GS-441524 is metabolized to the pharmacologically active GS-441524 triphosphate (TP) less efficiently than RDV, which correlates with a lower in vitro SARS-CoV-2 antiviral activity. In vivo , African green monkeys (AGM) orally dosed with GS-441524 yielded low plasma levels due to limited oral bioavailability of <10%. When GS-441524 was delivered via intravenous (IV) administration, although plasma concentrations of GS-441524 were significantly higher, lung TP levels were lower than observed from IV RDV. To determine the required systemic exposure of GS-441524 associated with in vivo antiviral efficacy, SARS-CoV-2 infected AGMs were treated with a once-daily IV dose of either 7.5 or 20 mg/kg GS-441524 or IV RDV for 5 days and compared to vehicle control. Despite the reduced lung TP formation compared to IV dosing of RDV, daily treatment with IV GS-441524 resulted in dose-dependent efficacy, with the 20 mg/kg GS-441524 treatment resulting in significant reductions of SARS-CoV-2 replication in the lower respiratory tract of infected animals. These findings demonstrate the in vivo SARS-CoV-2 antiviral efficacy of GS-441524 and support evaluation of its orally bioavailable prodrugs as potential therapies for COVID-19. • GS-441524 and remdesivir both inhibit SARS-CoV-2 in primary human lung cells. • Limited oral bioavailability of GS-441524 in primates necessitates IV administration to yield high plasma concentrations. • Intravenous GS-441524 and remdesivir both reduce SARS-CoV-2 viral burden AGM model. • Antiviral efficacy of GS-441524 suggests prodrugs that increase oral bioavailability are desirable to identify and develop. [ABSTRACT FROM AUTHOR]
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- 2022
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25. Discoveryof the First C-Nucleoside HCV Polymerase Inhibitor(GS-6620) with Demonstrated Antiviral Response in HCV Infected Patients.
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Cho, Aesop, Zhang, Lijun, Xu, Jie, Lee, Rick, Butler, Thomas, Metobo, Sammy, Aktoudianakis, Vangelis, Lew, Willard, Ye, Hong, Clarke, Michael, Doerffler, Edward, Byun, Daniel, Wang, Ting, Babusis, Darius, Carey, Anne C., German, Polina, Sauer, Dorothea, Zhong, Weidong, Rossi, Stephen, and Fenaux, Martijn
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- 2014
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26. GS-9219/VDC-1101 - a prodrug of the acyclic nucleotide PMEG has antitumor activity in spontaneous canine multiple myeloma.
- Author
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Thamm, Douglas H., Vail, David M., Kurzman, Ilene D., Babusis, Darius, Ray, Adrian S., Sousa-Powers, Noel, and Tumas, Daniel B.
- Subjects
MULTIPLE myeloma ,B cell lymphoma ,CELL culture ,CULTURES (Biology) ,BLOOD plasma - Abstract
Background Multiple myeloma (MM) is an important human and canine cancer for which novel therapies remain necessary. VDC-1101 (formerly GS-9219), a novel double prodrug of the antiproliferative nucleotide analog 9-(2-phosphonylmethoxyethyl) guanine (PMEG), possesses potent cytotoxic activity in vitro in human lymphoblasts and leukemia cell lines and in vivo in spontaneous canine lymphoma. Given the similarity in lineage between lymphoma and MM, we hypothesized that VDC-1101 would be active against MM. Results We evaluated the in vitro antiproliferative effects of VDC-1101 against 3 human MM cell lines, and we performed a phase-II clinical trial in 14 dogs with spontaneous MM. Each dog was treated with a maximum of 6 doses of VDC-1101 monotherapy over 10-15 weeks. Dosedependent antiproliferative activity was observed in all evaluated cell lines. Major antitumor responses (reduction of serum paraprotein and resolution of hypercalcemia, peripheral cytopenias and bone marrow plasmacytosis) were observed in 9 of 11 evaluable dogs for a median of 172 days, including a durable stringent complete response (>1047 days) in a dog with melphalan-refractory disease. 2 dogs were euthanized due to presumed pulmonary fibrosis; there were no other dose-limiting toxicities encountered. Conclusions In conclusion, VDC-1101 has significant anti-tumor activity at well-tolerated doses in spontaneous canine MM. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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27. Sensitivity of Mitochondrial Transcription and Resistance of RNA Polymerase II Dependent Nuclear Transcription to Antiviral Ribonucleosides.
- Author
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Arnold, Jamie J., Sharma, Suresh D., Feng, Joy Y., Ray, Adrian S., Smidansky, Eric D., Kireeva, Maria L., Cho, Aesop, Perry, Jason, Vela, Jennifer E., Yeojin Park, Yili Xu, Yang Tian, Babusis, Darius, Barauskus, Ona, Peterson, Blake R., Gnatt, Averell, Kashlev, Mikhail, Weidong Zhong, and Cameron, Craig E.
- Subjects
MITOCHONDRIA ,ORGANELLES ,RNA polymerases ,TRANSFERASES ,RIBONUCLEOSIDES - Abstract
Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C- methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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28. Effects of the Preservative Purite® on the Bioavailability of Brimonidine in the Aqueous Humor of Rabbits.
- Author
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Dong, Jennifer Q., Babusis, Darius M., Welty, Devin F., Acheampong, Andrew A., Tang-Liu, Diane, and Whitcup, Scott M.
- Subjects
- *
ANTERIOR chamber (Eye) , *AQUEOUS humor , *BIOAVAILABILITY , *LABORATORY rabbits , *EUROPEAN rabbit , *BODY fluids - Abstract
Purpose: To determine aqueous humor concentrations of brimonidine given the following ophthalmic formulations in female New Zealand White Rabbits: (1) BAK-preserved brimonidine tartrate 0.20% at a pH of 6.4; (2) BAK-preserved brimonidine tartrate 0.15% at a pH of 6.4, and (3) Purite®-preserved brimonidine tartrate 0.15% at a pH of 7.3. Methods: Eighteen (18) animals were given a 35- L drop of formulation into each eye. Aqueous humor samples were collected at 9 time points over 8 hours. Brimonidine concentrations were quantified using LC-MS/MS. Results: The Cmax was achieved between 0.33–0.67 hours postdosing for all 3 formulations. Mean Cmax after Purite-preserved brimonidine tartrate 0.15% was 88% higher than that after BAK-preserved brimonidine tartrate 0.15% (p = 0.040), and 44% higher than that after BAK-preserved brimonidine tartrate 0.20% (p = 0.0784). AUC0–3 hr values were comparable for all 3 formulations. Conclusions : Purite-preserved brimonidine tartrate 0.15% produced higher peak concentrations than BAK-preserved brimonidine tartrate 0.15%. It also had a concentration that was comparable to BAK-preserved brimonidine tartrate 0.20%. The differences in safety may result from the change in preservative. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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29. Pomalidomide Shows Significant Therapeutic Activity against CNS Lymphoma with a Major Impact on the Tumor Microenvironment in Murine Models.
- Author
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Li, Zhimin, Qiu, Yushi, Personett, David, Huang, Peng, Edenfield, Brandy, Katz, Jason, Babusis, Darius, Tang, Yang, Shirely, Michael A., Moghaddam, Mehran F., Copland, John A., and Tun, Han W.
- Subjects
LYMPHOMAS ,TREATMENT of central nervous system cancer ,DISEASE progression ,IMMUNOREGULATION ,ANTINEOPLASTIC agents ,LABORATORY mice - Abstract
Primary CNS lymphoma carries a poor prognosis. Novel therapeutic agents are urgently needed. Pomalidomide (POM) is a novel immunomodulatory drug with anti-lymphoma activity. CNS pharmacokinetic analysis was performed in rats to assess the CNS penetration of POM. Preclinical evaluation of POM was performed in two murine models to assess its therapeutic activity against CNS lymphoma. The impact of POM on the CNS lymphoma immune microenvironment was evaluated by immunohistochemistry and immunofluorescence. In vitro cell culture experiments were carried out to further investigate the impact of POM on the biology of macrophages. POM crosses the blood brain barrier with CNS penetration of ~ 39%. Preclinical evaluations showed that it had significant therapeutic activity against CNS lymphoma with significant reduction in tumor growth rate and prolongation of survival, that it had a major impact on the tumor microenvironment with an increase in macrophages and natural killer cells, and that it decreased M2-polarized tumor-associated macrophages and increased M1-polarized macrophages when macrophages were evaluated based on polarization status. In vitro studies using various macrophage models showed that POM converted the polarization status of IL4-stimulated macrophages from M2 to M1, that M2 to M1 conversion by POM in the polarization status of lymphoma-associated macrophages is dependent on the presence of NK cells, that POM induced M2 to M1 conversion in the polarization of macrophages by inactivating STAT6 signaling and activating STAT1 signaling, and that POM functionally increased the phagocytic activity of macrophages. Based on our findings, POM is a promising therapeutic agent for CNS lymphoma with excellent CNS penetration, significant preclinical therapeutic activity, and a major impact on the tumor microenvironment. It can induce significant biological changes in tumor-associated macrophages, which likely play a major role in its therapeutic activity against CNS lymphoma. POM should be further evaluated in clinical trials. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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30. Tenofovir alafenamide (TAF) does not deplete mitochondrial DNA in human T-cell lines at intracellular concentrations exceeding clinically relevant drug exposures.
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Stray, Kirsten M., Park, Yeojin, Babusis, Darius, Callebaut, Christian, Cihlar, Tomas, Ray, Adrian S., and Perron, Michel
- Subjects
- *
HIV infections , *THERAPEUTICS , *TENOFOVIR , *REVERSE transcriptase inhibitors , *CELL lines , *MITOCHONDRIAL DNA - Abstract
HIV-infected patients treated with certain nucleoside reverse transcriptase inhibitors (NRTIs) have experienced adverse effects due to drug-related mitochondrial toxicity. Tenofovir alafenamide (TAF) is a novel prodrug of the NRTI tenofovir (TFV) with an improved safety profile compared to tenofovir disoproxil fumarate (TDF). Prior in vitro studies have demonstrated that the parent nucleotide TFV has no significant effects on mtDNA synthesis. This study investigated whether clinically relevant TAF and TDF exposures affect mtDNA content in human lymphocytes. First, activated or resting peripheral blood mononuclear cells (PBMCs), as well as MT-2 and Jurkat T-cell lines, were continuously treated with ddC for 10 days to establish their susceptibility to mtDNA depletion. PBMCs had low sensitivity to NRTI-mediated mtDNA depletion in vitro . In contrast, ddC treatment of rapidly dividing MT-2 and Jurkat cells resulted in a dose-dependent decrease in mtDNA. Therefore, these two T-cell lines were selected for evaluating TAF and TDF treatment effects. MT-2 and Jurkat cells were pulse-treated with TAF or TDF every 24 h for 10 days to mimic pharmacologically relevant drug exposures. Pulse treatment of cells with 3.3 μM TAF or 1.1 μM TDF for 10 days resulted in 2- to 7-fold greater steady-state intracellular TFV-diphosphate (TFV-DP) levels than those observed clinically in TAF- or TDF-treated patients. At these concentrations, no significant TAF- (106.7% and 84.1% of control; p = 0.77 and 0.12 for MT-2 and Jurkat, respectively) or TDF- (100.6% and 91.0% of control; p = 0.91 and 0.37, respectively) associated reduction in mtDNA content was observed compared with untreated control cells. This study demonstrates that, despite delivering higher intracellular levels of TFV-DP than TDF, TAF does not inhibit mtDNA synthesis in vitro at concentrations exceeding the clinically relevant intracellular drug exposures. Thus, TAF has a low potential for mitochondrial toxicity in T-cells of HIV-infected patients. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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31. Subcutaneous remdesivir administration prevents interstitial pneumonia in rhesus macaques inoculated with SARS-CoV-2.
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Williamson, Brandi N., Pérez-Pérez, Lizzette, Schwarz, Benjamin, Feldmann, Friederike, Holbrook, Myndi G., Singh, Manmeet, Lye, Diane S., Babusis, Darius, Subramanian, Raju, Haddock, Elaine, Okumura, Atsushi, Hanley, Patrick W., Lovaglio, Jamie, Bosio, Catharine M., Porter, Danielle P., Cihlar, Tomas, Mackman, Richard L., Saturday, Greg, and de Wit, Emmie
- Subjects
- *
MACAQUES , *RHESUS monkeys , *PULMONARY fibrosis , *REMDESIVIR , *SARS-CoV-2 , *RESPIRATORY diseases - Abstract
The utility of remdesivir treatment in COVID-19 patients is currently limited by the necessity to administer this antiviral intravenously, which has generally limited its use to hospitalized patients. Here, we tested a novel, subcutaneous formulation of remdesivir in the rhesus macaque model of SARS-CoV-2 infection that was previously used to establish the efficacy of remdesivir against this virus in vivo. Compared to vehicle-treated animals, macaques treated with subcutaneous remdesivir from 12 h through 6 days post inoculation showed reduced signs of respiratory disease, a reduction of virus replication in the lower respiratory tract, and an absence of interstitial pneumonia. Thus, early subcutaneous administration of remdesivir can protect from lower respiratory tract disease caused by SARS-CoV-2. • Remdesivir is a nucleotide analog prodrug with broad-spectrum antiviral activity shown to be effective against SARS-CoV-2. • The utility of remdesivir treatment in COVID-19 patients is limited by the necessity to administer it intravenously. • The efficacy of a subcutaneous formulation of remdesivir was tested in the rhesus macaque model of SARS-CoV-2 infection. • Subcutaneous remdesivir reduced respiratory signs, reduced replication in the lungs, and prevented interstitial pneumonia. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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32. Characterization of the Cynomolgus Macaque Model of Marburg Virus Disease and Assessment of Timing for Therapeutic Treatment Testing.
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Zumbrun EE, Garvey CB, Wells JB, Lynn GC, Van Tongeren S, Steffens JT, Wetzel KS, Gomba LM, O'Brien KA, Rossi FD, Zeng X, Lee ED, Raymond JLW, Hoffman DA, Jay AN, Brown ES, Kallgren PA, Norris SL, Cantey-Kiser J, Kudiya H, Arthur C, Blair C, Babusis D, Chu VC, Singh B, Bannister R, Porter DP, Cihlar T, and Dye JM
- Subjects
- Humans, Animals, Macaca fascicularis, Viremia, Liver, Marburg Virus Disease, Marburgvirus
- Abstract
Marburg virus (MARV) causes severe disease and high mortality in humans. The objective of this study was to characterize disease manifestations and pathogenesis in cynomolgus macaques exposed to MARV. The results of this natural history study may be used to identify features of MARV disease useful in defining the ideal treatment initiation time for subsequent evaluations of investigational therapeutics using this model. Twelve cynomolgus macaques were exposed to a target dose of 1000 plaque-forming units MARV by the intramuscular route, and six control animals were mock-exposed. The primary endpoint of this study was survival to Day 28 post-inoculation (PI). Anesthesia events were minimized with the use of central venous catheters for periodic blood collection, and temperature and activity were continuously monitored by telemetry. All mock-exposed animals remained healthy for the duration of the study. All 12 MARV-exposed animals (100%) became infected, developed illness, and succumbed on Days 8-10 PI. On Day 4 PI, 11 of the 12 MARV-exposed animals had statistically significant temperature elevations over baseline. Clinically observable signs of MARV disease first appeared on Day 5 PI, when 6 of the 12 animals exhibited reduced responsiveness. Ultimately, systemic inflammation, coagulopathy, and direct cytopathic effects of MARV all contributed to multiorgan dysfunction, organ failure, and death or euthanasia of all MARV-exposed animals. Manifestations of MARV disease, including fever, systemic viremia, lymphocytolysis, coagulopathy, and hepatocellular damage, could be used as triggers for initiation of treatment in future therapeutic efficacy studies.
- Published
- 2023
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33. Efficacy of the oral nucleoside prodrug GS-5245 (Obeldesivir) against SARS-CoV-2 and coronaviruses with pandemic potential.
- Author
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Martinez DR, Moreira FR, Zweigart MR, Gully KL, De la Cruz G, Brown AJ, Adams LE, Catanzaro N, Yount B, Baric TJ, Mallory ML, Conrad H, May SR, Dong S, Scobey DT, Montgomery SA, Perry J, Babusis D, Barrett KT, Nguyen AH, Nguyen AQ, Kalla R, Bannister R, Bilello JP, Feng JY, Cihlar T, Baric RS, Mackman RL, Schäfer A, and Sheahan TP
- Abstract
Despite the wide availability of several safe and effective vaccines that can prevent severe COVID-19 disease, the emergence of SARS-CoV-2 variants of concern (VOC) that can partially evade vaccine immunity remains a global health concern. In addition, the emergence of highly mutated and neutralization-resistant SARS-CoV-2 VOCs such as BA.1 and BA.5 that can partially or fully evade (1) many therapeutic monoclonal antibodies in clinical use underlines the need for additional effective treatment strategies. Here, we characterize the antiviral activity of GS-5245, Obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved RNA-dependent viral RNA polymerase (RdRp). Importantly, we show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-related Bat-CoV RsSHC014, Middle East Respiratory Syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant in vitro and highly effective as antiviral therapy in mouse models of SARS-CoV, SARS-CoV-2 (WA/1), MERS-CoV and Bat-CoV RsSHC014 pathogenesis. In all these models of divergent coronaviruses, we observed protection and/or significant reduction of disease metrics such as weight loss, lung viral replication, acute lung injury, and degradation in pulmonary function in GS-5245-treated mice compared to vehicle controls. Finally, we demonstrate that GS-5245 in combination with the main protease (M
pro ) inhibitor nirmatrelvir had increased efficacy in vivo against SARS-CoV-2 compared to each single agent. Altogether, our data supports the continuing clinical evaluation of GS-5245 in humans infected with COVID-19, including as part of a combination antiviral therapy, especially in populations with the most urgent need for more efficacious and durable interventions., Competing Interests: DECLARATION OF INTERESTS These authors are employees of Gilead Sciences and hold stock in Gilead Sciences: D.B., A.N., K.T.B., R.B, J.P.B., J.Y.F., T.C., R.L.M.- Published
- 2023
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34. Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice.
- Author
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Schäfer A, Martinez DR, Won JJ, Moreira FR, Brown AJ, Gully KL, Kalla R, Chun K, Du Pont V, Babusis D, Tang J, Murakami E, Subramanian R, Barrett KT, Bleier BJ, Bannister R, Feng JY, Bilello JP, Cihlar T, Mackman RL, Montgomery SA, Baric RS, and Sheahan TP
- Abstract
The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans.
- Published
- 2021
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35. Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells.
- Author
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Li R, Liclican A, Xu Y, Pitts J, Niu C, Zhang J, Kim C, Zhao X, Soohoo D, Babusis D, Yue Q, Ma B, Murray BP, Subramanian R, Xie X, Zou J, Bilello JP, Li L, Schultz BE, Sakowicz R, Smith BJ, Shi PY, Murakami E, and Feng JY
- Subjects
- Adenosine Monophosphate analogs & derivatives, Alanine analogs & derivatives, Antiviral Agents pharmacology, Humans, Lung, Nerve Tissue Proteins, SARS-CoV-2, COVID-19 Drug Treatment
- Abstract
Remdesivir (RDV; GS-5734, Veklury), the first FDA-approved antiviral to treat COVID-19, is a single-diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which, in turn, acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (i) bioinformatic analysis of nucleoside/nucleotide metabolic enzyme mRNA expression using public human tissue and lung single-cell bulk mRNA sequence (RNA-seq) data sets, (ii) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells, (iii) biochemical studies on the catalytic rate of key enzymes, (iv) effects of specific enzyme inhibitors on the GS-443902 formation, and (v) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate MetX, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19 but also enable efficient intracellular metabolism of RDV and its MetX to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.
- Published
- 2021
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36. Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C -Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.
- Author
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Mackman RL, Hui HC, Perron M, Murakami E, Palmiotti C, Lee G, Stray K, Zhang L, Goyal B, Chun K, Byun D, Siegel D, Simonovich S, Du Pont V, Pitts J, Babusis D, Vijjapurapu A, Lu X, Kim C, Zhao X, Chan J, Ma B, Lye D, Vandersteen A, Wortman S, Barrett KT, Toteva M, Jordan R, Subramanian R, Bilello JP, and Cihlar T
- Subjects
- Adenosine Monophosphate pharmacology, Alanine pharmacology, Animals, Antiviral Agents chemistry, Antiviral Agents pharmacokinetics, Caco-2 Cells, Cells, Cultured, Chlorocebus aethiops, Disease Models, Animal, Dogs, Drug Evaluation, Preclinical methods, Epithelial Cells virology, Humans, Macaca fascicularis, Male, Prodrugs chemistry, Prodrugs pharmacokinetics, Rats, Sprague-Dawley, Respiratory Syncytial Virus Infections virology, Structure-Activity Relationship, Tissue Distribution, Tubercidin analogs & derivatives, Tubercidin chemistry, Viral Load, Rats, Adenosine Monophosphate analogs & derivatives, Alanine analogs & derivatives, Antiviral Agents pharmacology, Prodrugs pharmacology, Respiratory Syncytial Virus Infections drug therapy, Respiratory Syncytial Virus, Human drug effects
- Abstract
A discovery program targeting respiratory syncytial virus (RSV) identified C -nucleoside 4 (RSV A2 EC
50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir ( 1 , GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1 - NTP , and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4 . A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.- Published
- 2021
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37. Off-Target In Vitro Profiling Demonstrates that Remdesivir Is a Highly Selective Antiviral Agent.
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Xu Y, Barauskas O, Kim C, Babusis D, Murakami E, Kornyeyev D, Lee G, Stepan G, Perron M, Bannister R, Schultz BE, Sakowicz R, Porter D, Cihlar T, and Feng JY
- Subjects
- Adenosine Monophosphate chemistry, Adenosine Monophosphate pharmacology, Alanine chemistry, Alanine pharmacology, Antiviral Agents chemistry, COVID-19 virology, Cell Line, Epithelial Cells drug effects, Humans, Inhibitory Concentration 50, Mitochondria drug effects, Primary Cell Culture, Adenosine Monophosphate analogs & derivatives, Alanine analogs & derivatives, Antiviral Agents pharmacology, SARS-CoV-2 drug effects, COVID-19 Drug Treatment
- Abstract
Remdesivir (RDV, GS-5734), the first FDA-approved antiviral for the treatment of COVID-19, is a single diastereomer monophosphoramidate prodrug of an adenosine analogue. It is intracellularly metabolized into the active triphosphate form, which in turn acts as a potent and selective inhibitor of multiple viral RNA polymerases. RDV has broad-spectrum activity against members of the coronavirus family, such as SARS-CoV-2, SARS-CoV, and MERS-CoV, as well as filoviruses and paramyxoviruses. To assess the potential for off-target toxicity, RDV was evaluated in a set of cellular and biochemical assays. Cytotoxicity was evaluated in a set of relevant human cell lines and primary cells. In addition, RDV was evaluated for mitochondrial toxicity under aerobic and anaerobic metabolic conditions, and for the effects on mitochondrial DNA content, mitochondrial protein synthesis, cellular respiration, and induction of reactive oxygen species. Last, the active 5'-triphosphate metabolite of RDV, GS-443902, was evaluated for potential interaction with human DNA and RNA polymerases. Among all of the human cells tested under 5 to 14 days of continuous exposure, the 50% cytotoxic concentration (CC
50 ) values of RDV ranged from 1.7 to >20 μM, resulting in selectivity indices (SI, CC50 /EC50 ) from >170 to 20,000, with respect to RDV anti-SARS-CoV-2 activity (50% effective concentration [EC50 ] of 9.9 nM in human airway epithelial cells). Overall, the cellular and biochemical assays demonstrated a low potential for RDV to elicit off-target toxicity, including mitochondria-specific toxicity, consistent with the reported clinical safety profile., (Copyright © 2021 Xu et al.)- Published
- 2021
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38. Remdesivir Inhibits SARS-CoV-2 in Human Lung Cells and Chimeric SARS-CoV Expressing the SARS-CoV-2 RNA Polymerase in Mice.
- Author
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Pruijssers AJ, George AS, Schäfer A, Leist SR, Gralinksi LE, Dinnon KH 3rd, Yount BL, Agostini ML, Stevens LJ, Chappell JD, Lu X, Hughes TM, Gully K, Martinez DR, Brown AJ, Graham RL, Perry JK, Du Pont V, Pitts J, Ma B, Babusis D, Murakami E, Feng JY, Bilello JP, Porter DP, Cihlar T, Baric RS, Denison MR, and Sheahan TP
- Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the novel viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV) potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC
50 = 0.01 μM). Weaker activity is observed in Vero E6 cells (EC50 = 1.65 μM) because of their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase of SARS-CoV-2. In mice infected with the chimeric virus, therapeutic RDV administration diminishes lung viral load and improves pulmonary function compared with vehicle-treated animals. These data demonstrate that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19., Competing Interests: Declaration of Interests The authors affiliated with Gilead Sciences, Inc. are employees of the company and may own company stock., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2020
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39. Species differences in liver accumulation and metabolism of nucleotide prodrug sofosbuvir.
- Author
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Wang T, Babusis D, Park Y, Niu C, Kim C, Zhao X, Lu B, Ma B, Muench RC, Sperger D, Ray AS, and Murakami E
- Subjects
- Animals, Dogs, Humans, Macaca fascicularis, Male, Mice, Mice, Inbred C57BL, Molecular Conformation, Prodrugs chemistry, Prodrugs pharmacokinetics, Rats, Rats, Sprague-Dawley, Sofosbuvir chemistry, Sofosbuvir pharmacokinetics, Liver chemistry, Liver metabolism, Prodrugs metabolism, Sofosbuvir metabolism
- Abstract
Sofosbuvir (SOF) is a nucleotide prodrug which has been used as a backbone for the clinical treatment of hepatitis C viral infection. Because sofosbuvir undergoes complex first pass metabolism, including metabolic activation to form its pharmacologically active triphosphate (GS-331007-TP) to inhibit the viral RNA polymerase in the liver, it is difficult to project the human dose for clinical evaluation based on preclinical data. Selecting an appropriate animal model for drug exposure in the target tissue is challenging due to differences in absorption, stability, hepatic uptake, and intracellular activation across species. Efficient liver delivery has been established in human liver following administration in a clinical trial of patients receiving sofosbuvir prior to liver transplantation. Using the clinical liver exposure as a benchmark, we assessed and compared the pharmacokinetic profile in mouse, rat, hamster, dog and monkey. Liver accumulation was also assessed in the PXB mouse model in which the liver is mostly populated with human hepatocytes. At human equivalent dose, the hepatic concentrations of GS-331007-TP in dog and PXB mouse were comparable to those observed in the human livers. In these species, high and sustained levels of GS-331007-TP were observed in both primary hepatocytes in vitro and the liver in vivo., Competing Interests: Declaration of competing interest All the authors are current or former employee of Gilead Sciences., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2020
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40. Remdesivir potently inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice.
- Author
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Pruijssers AJ, George AS, Schäfer A, Leist SR, Gralinksi LE, Dinnon KH, Yount BL, Agostini ML, Stevens LJ, Chappell JD, Lu X, Hughes TM, Gully K, Martinez DR, Brown AJ, Graham RL, Perry JK, Du Pont V, Pitts J, Ma B, Babusis D, Murakami E, Feng JY, Bilello JP, Porter DP, Cihlar T, Baric RS, Denison MR, and Sheahan TP
- Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC
50 = 0.01 μM). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 μM) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo , supporting its further clinical testing for treatment of COVID-19.- Published
- 2020
- Full Text
- View/download PDF
41. Nucleotide Prodrug Containing a Nonproteinogenic Amino Acid To Improve Oral Delivery of a Hepatitis C Virus Treatment.
- Author
-
Feng JY, Wang T, Park Y, Babusis D, Birkus G, Xu Y, Voitenleitner C, Fenaux M, Yang H, Eng S, Tirunagari N, Kirschberg T, Cho A, and Ray AS
- Subjects
- Adenosine analogs & derivatives, Administration, Oral, Alanine, Animals, Antiviral Agents administration & dosage, Antiviral Agents pharmacokinetics, Caco-2 Cells, Cells, Cultured, Dogs, Hepacivirus drug effects, Hepatitis C virology, Humans, Male, Nucleotides administration & dosage, Nucleotides pharmacokinetics, Prodrugs administration & dosage, Prodrugs pharmacokinetics, Rats, Triazines administration & dosage, Triazines pharmacokinetics, Virus Replication drug effects, Antiviral Agents therapeutic use, Hepacivirus pathogenicity, Nucleotides therapeutic use, Prodrugs therapeutic use, Triazines therapeutic use
- Abstract
Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2'- C -methyl-1'-cyano-7-deaza-adenosine C -nucleotide analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection. However, the prodrug selected for clinical development, GS-6620, required a high dose for meaningful efficacy and had unacceptable variability due to poor oral absorption as a result of suboptimal solubility, intestinal metabolism, and efflux transport. While obtaining clinical proof of concept for the nucleotide analog, a more effective prodrug strategy would be necessary for clinical utility. Here, we report an alternative prodrug of the same nucleoside analog identified to address liabilities of GS-6620. A phosphoramidate prodrug containing the nonproteinogenic amino acid methylalanine, an isopropyl ester and phenol in the ( S ) conformation at phosphorous, GS2, was found to have improved solubility, intestinal stability, and hepatic activation. GS2 is a more selective substrate for hepatically expressed carboxyl esterase 1 (CES1) and is resistant to hydrolysis by more widely expressed hydrolases, including cathepsin A (CatA) and CES2. Unlike GS-6620, GS2 was not cleaved by intestinally expressed CES2 and, as a result, was stable in intestinal extracts. Levels of liver triphosphate following oral administration of GS2 in animals were higher than those of GS-6620, even when administered under optimal conditions for GS-6620 absorption. Combined, these properties suggest that GS2 will have better oral absorption in the clinic when administered in a solid dosage form and the potential to extend the clinical proof of concept obtained with GS-6620., (Copyright © 2018 Feng et al.)
- Published
- 2018
- Full Text
- View/download PDF
42. Sofosbuvir and Ribavirin Liver Pharmacokinetics in Patients Infected with Hepatitis C Virus.
- Author
-
Babusis D, Curry MP, Kirby B, Park Y, Murakami E, Wang T, Mathias A, Afdhal N, McHutchison JG, and Ray AS
- Subjects
- Aged, Female, Hepatitis C metabolism, Hepatitis C virology, Humans, Male, Mass Spectrometry, Middle Aged, Antiviral Agents pharmacokinetics, Antiviral Agents therapeutic use, Hepacivirus drug effects, Hepacivirus pathogenicity, Hepatitis C drug therapy, Liver metabolism, Liver virology, Ribavirin pharmacokinetics, Ribavirin therapeutic use, Sofosbuvir pharmacokinetics, Sofosbuvir therapeutic use
- Abstract
Sofosbuvir and ribavirin exert their anti-hepatitis C virus (anti-HCV) activity following metabolic activation in the liver. However, intrahepatic concentrations of the pharmacologically active nucleotide metabolites in humans are poorly characterized due to the inaccessibility of tissue and technical challenges with measuring nucleotide levels. A clinical study assessing the efficacy of sofosbuvir and ribavirin administered prior to liver transplantation to prevent HCV recurrence provided a unique opportunity to quantify nucleotide concentrations in human liver. We analyzed nucleotides using high-performance liquid chromatography coupled to tandem mass spectrometry in liver tissue from 30 HCV-infected patients with hepatocellular carcinoma who were administered sofosbuvir (400 mg/day) and ribavirin (1,000 to 1,200 mg/day) for 3 to 52 weeks prior to liver transplantation. Median total hepatic metabolite concentrations (the sum of nucleoside and mono-, di-, and triphosphates) were 77.1 μM for sofosbuvir and 361 μM for ribavirin in patients on therapy at the time of transplantation. Ribavirin and sofosbuvir efficiently loaded the liver, with total hepatic metabolite concentrations exceeding maximal levels in plasma by approximately 30-fold. Ribavirin metabolite levels suggest that its monophosphate is in great excess of its inhibition constant for IMP dehydrogenase and that its triphosphate is approaching the binding constant for incorporation by the HCV NS5B RNA-dependent RNA polymerase. In accordance with the potent antiviral activity of sofosbuvir, these results demonstrate that the liver triphosphate levels achieved following sofosbuvir administration greatly exceed the inhibition constant for HCV NS5B. In conclusion, this study expands the quantitative understanding of the pharmacology of sofosbuvir and ribavirin by establishing efficient hepatic delivery in the clinic. (This study has been registered at ClinicalTrials.gov under identifier NCT01559844.)., (Copyright © 2018 American Society for Microbiology.)
- Published
- 2018
- Full Text
- View/download PDF
43. Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus.
- Author
-
Feng JY, Xu Y, Barauskas O, Perry JK, Ahmadyar S, Stepan G, Yu H, Babusis D, Park Y, McCutcheon K, Perron M, Schultz BE, Sakowicz R, and Ray AS
- Subjects
- Cell Line, DNA Polymerase gamma, DNA-Directed DNA Polymerase genetics, DNA-Directed RNA Polymerases genetics, Deoxycytidine analogs & derivatives, Deoxycytidine pharmacology, Guanosine Monophosphate analogs & derivatives, Guanosine Monophosphate pharmacology, Humans, Mitochondria genetics, Mitochondria metabolism, Nucleosides pharmacology, Oxygen Consumption drug effects, Protein Biosynthesis drug effects, RNA genetics, RNA, Mitochondrial, Sofosbuvir pharmacology, Transcription, Genetic drug effects, Transcription, Genetic genetics, Virus Replication drug effects, Antiviral Agents pharmacology, DNA-Directed RNA Polymerases drug effects, Hepacivirus drug effects, Hepatitis C, Chronic drug therapy, Mitochondria drug effects
- Abstract
Toxicity has emerged during the clinical development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clinically relevant HCV NI were characterized in biochemical and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4'-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2'-C-methyl guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concentrations, followed by general cytotoxicity. In contrast, NI containing multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2'-deoxynucleotide analogs, there is an association between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clinical testing., (Copyright © 2016 Feng et al.)
- Published
- 2015
- Full Text
- View/download PDF
44. Implications of efficient hepatic delivery by tenofovir alafenamide (GS-7340) for hepatitis B virus therapy.
- Author
-
Murakami E, Wang T, Park Y, Hao J, Lepist EI, Babusis D, and Ray AS
- Subjects
- Adenine metabolism, Adenine pharmacokinetics, Adenine pharmacology, Alanine, Animals, Cells, Cultured, Dogs, Hepatitis B virus pathogenicity, Hepatocytes drug effects, Humans, Liver drug effects, Liver metabolism, Male, Organophosphates metabolism, Tenofovir analogs & derivatives, Adenine analogs & derivatives, Hepatitis B virus drug effects, Hepatocytes metabolism
- Abstract
Tenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytes in vitro and in dogs in vivo were evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a half-life of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with the in vitro data, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
- Full Text
- View/download PDF
45. Discovery of the first C-nucleoside HCV polymerase inhibitor (GS-6620) with demonstrated antiviral response in HCV infected patients.
- Author
-
Cho A, Zhang L, Xu J, Lee R, Butler T, Metobo S, Aktoudianakis V, Lew W, Ye H, Clarke M, Doerffler E, Byun D, Wang T, Babusis D, Carey AC, German P, Sauer D, Zhong W, Rossi S, Fenaux M, McHutchison JG, Perry J, Feng J, Ray AS, and Kim CU
- Subjects
- Animals, Antiviral Agents pharmacokinetics, Antiviral Agents pharmacology, Dogs, Enzyme Inhibitors pharmacokinetics, Enzyme Inhibitors pharmacology, Hepatitis C enzymology, Hepatitis C virology, Humans, Nucleosides chemistry, Organophosphorus Compounds chemistry, Rats, Viral Load, Antiviral Agents therapeutic use, Enzyme Inhibitors therapeutic use, Hepacivirus enzymology, Hepatitis C drug therapy, Nucleosides pharmacology, Organophosphorus Compounds pharmacology, Viral Nonstructural Proteins antagonists & inhibitors
- Abstract
Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clinical development thus far have all been N-nucleoside derivatives. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-methyl 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compounds showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clinical development candidate GS-6620. In a phase I clinical study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.
- Published
- 2014
- Full Text
- View/download PDF
46. Metabolism and pharmacokinetics of the anti-hepatitis C virus nucleotide prodrug GS-6620.
- Author
-
Murakami E, Wang T, Babusis D, Lepist EI, Sauer D, Park Y, Vela JE, Shih R, Birkus G, Stefanidis D, Kim CU, Cho A, and Ray AS
- Subjects
- Administration, Oral, Animals, Antiviral Agents pharmacology, Caco-2 Cells, Cell Line, Cricetinae, Dogs, Hepacivirus drug effects, Humans, Macaca fascicularis, Male, Mesocricetus, Prodrugs pharmacology, Antiviral Agents pharmacokinetics, Prodrugs pharmacokinetics
- Abstract
The anti-hepatitis C virus nucleotide prodrug GS-6620 employs a double-prodrug approach, with l-alanine-isopropyl ester and phenol moieties attached to the 5'-phosphate that release the nucleoside monophosphate in hepatocytes and a 3'-isobutyryl ester added to improve permeability and oral bioavailability. Consistent with the stability found in intestinal homogenates, following oral administration, intact prodrug levels in blood plasma were the highest in dogs, followed by monkeys, and then were the lowest in hamsters. In contrast, liver levels of the triphosphate metabolite at the equivalent surface area-adjusted doses were highest in hamsters, followed by in dogs and monkeys. Studies in isolated primary hepatocytes suggest that relatively poor oral absorption in hamsters and monkeys was compensated for by relatively efficient hepatocyte activation. As intestinal absorption was found to be critical to the effectiveness of GS-6620 in nonclinical species, stomach pH, formulation, and food effect studies were completed in dogs. Consistent with in vitro absorption studies in Caco-2 cells, the absorption of GS-6620 was found to be complex and highly dependent on concentration. Higher rates of metabolism were observed at lower concentrations that were unable to saturate intestinal efflux transporters. In first-in-human clinical trials, the oral administration of GS-6620 resulted in poor plasma exposure relative to that observed in dogs and in large pharmacokinetic and pharmacodynamic variabilities. While a double-prodrug approach, including a 3'-isobutyryl ester, provided higher intrinsic intestinal permeability, this substitution appeared to be a metabolic liability, resulting in extensive intestinal metabolism and relatively poor oral absorption in humans.
- Published
- 2014
- Full Text
- View/download PDF
47. Metabolism and antiretroviral activity of tenofovir alafenamide in CD4+ T-cells and macrophages from demographically diverse donors.
- Author
-
Bam RA, Birkus G, Babusis D, Cihlar T, and Yant SR
- Subjects
- Adenine chemistry, Adenine metabolism, Adenine pharmacology, Alanine, Anti-HIV Agents metabolism, Anti-HIV Agents pharmacology, Antiviral Agents chemistry, Antiviral Agents metabolism, Antiviral Agents pharmacology, Cathepsin A metabolism, Cells, Cultured, Enzyme Activation drug effects, HIV-1 drug effects, Humans, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear metabolism, Tenofovir analogs & derivatives, Tissue Donors, Adenine analogs & derivatives, CD4-Positive T-Lymphocytes drug effects, CD4-Positive T-Lymphocytes metabolism, Macrophages drug effects, Macrophages metabolism
- Abstract
Background: Tenofovir alafenamide (TAF) is a novel investigational prodrug of tenofovir (TFV) that permits enhanced delivery of TFV into peripheral blood mononuclear cells (PBMCs) and lymphatic tissues. A critical step in the intracellular metabolic activation of TAF is mediated by the lysosomal protease cathepsin A (CatA). Here, we investigated CatA levels together with intracellular metabolism and antiretroviral activity of TAF in primary CD4+ T-lymphocytes (CD4s) and monocyte-derived macrophages (MDMs) isolated from a demographically diverse group of blood donors., Methods: CD4s and MDMs were prepared from fresh PBMCs. CatA levels were quantified in cell extracts by monitoring TAF hydrolysis using HPLC. Intracellular TAF metabolites were quantified by HPLC combined with mass spectrometry. Antiviral activities in activated CD4s and MDMs were determined using HIV-1 single-cycle reporter and p24 antigen production assays, respectively., Results: The levels of CatA and intracellular TAF metabolites differed minimally in CD4s and MDMs among 13 tested donors. TAF was >600-fold and 80-fold more potent than parent TFV in CD4s and MDMs, respectively, and its relative range of antiviral activity across all tested donors was comparable to that of other HIV-1 reverse transcriptase inhibitors, with mean ±sd (range) EC50 values of 11.0 ±3.4 (6.6-19.9) nM and 9.7 ±4.6 (2.5-15.7) nM in CD4s and MDMs, respectively., Conclusions: These results indicate consistent intracellular metabolism and antiretroviral potency of TAF in relevant target cells of HIV-1 infection across multiple donors of variable gender, age and ethnicity, supporting further clinical investigation of TAF.
- Published
- 2014
- Full Text
- View/download PDF
48. Mechanism for effective lymphoid cell and tissue loading following oral administration of nucleotide prodrug GS-7340.
- Author
-
Babusis D, Phan TK, Lee WA, Watkins WJ, and Ray AS
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Adenine administration & dosage, Adenine blood, Adenine pharmacokinetics, Administration, Oral, Alanine, Animals, Caco-2 Cells, Cathepsin A administration & dosage, Cathepsin A blood, Cathepsin A pharmacokinetics, Chromatography, Liquid, Dogs, Humans, Intestinal Absorption, Male, Prodrugs pharmacokinetics, Tandem Mass Spectrometry, Tenofovir analogs & derivatives, Adenine analogs & derivatives, Lymphocytes metabolism
- Abstract
GS-7340 is a prodrug of tenofovir (TFV) that more efficiently delivers TFV into lymphoid cells and tissues than the clinically used prodrug TFV disoproxil fumarate, resulting in higher antiviral potency at greatly reduced doses and lower systemic TFV exposure. First-pass extraction by the intestine and liver represents substantial barriers to the oral delivery of prodrugs designed for rapid intracellular hydrolysis. In order to understand how GS-7340 reduces first-pass clearance to be an effective oral prodrug, its permeability and stability were characterized in vitro and detailed pharmacokinetic studies were completed in dogs. GS-7340 showed concentration-dependent permeability through monolayers of caco-2 cells and dose-dependent oral bioavailability in dogs, increasing from 1.7% at 2 mg/kg to 24.7% at 20 mg/kg, suggesting saturable intestinal efflux transport. Taking into account a 65% hepatic extraction measured in portal vein cannulated dogs, high dose GS-7340 is nearly completely absorbed. Consistent with the proposed role of intestinal efflux transport, coadministration of low dose GS-7340 with a transport inhibitor substantially increased GS-7340 exposure. The result of effective oral absorption and efficient lymphoid cell loading was reflected in the high and persistent levels of the pharmacologically active metabolite, TFV diphosphate, in peripheral blood mononuclear cells following oral administration to dogs. In conclusion, GS-7340 reaches the systemic circulation to effectively load target cells by saturating intestinal efflux transporters, facilitated by its high solubility, and by maintaining sufficient stability in intestinal and hepatic tissue.
- Published
- 2013
- Full Text
- View/download PDF
49. Compared to subcutaneous tenofovir, oral tenofovir disoproxyl fumarate administration preferentially concentrates the drug into gut-associated lymphoid cells in simian immunodeficiency virus-infected macaques.
- Author
-
Van Rompay KK, Babusis D, Abbott Z, Geng Y, Jayashankar K, Johnson JA, Lipscomb J, Heneine W, Abel K, and Ray AS
- Subjects
- Adenine administration & dosage, Adenine pharmacokinetics, Administration, Oral, Animals, Antiviral Agents administration & dosage, Drug Administration Schedule, Injections, Subcutaneous, Intestines chemistry, Intestines virology, Lymphocytes chemistry, Lymphocytes virology, Macaca mulatta, Male, Organophosphonates administration & dosage, RNA, Viral biosynthesis, Simian Acquired Immunodeficiency Syndrome virology, Simian Immunodeficiency Virus physiology, Tenofovir, Treatment Outcome, Adenine analogs & derivatives, Antiviral Agents pharmacokinetics, Organophosphonates pharmacokinetics, RNA, Viral antagonists & inhibitors, Simian Acquired Immunodeficiency Syndrome drug therapy, Simian Immunodeficiency Virus drug effects
- Abstract
To compare tissue-based pharmacokinetics and efficacy of oral tenofovir disoproxyl fumarate (TDF) versus subcutaneous tenofovir (TFV), macaques were treated for 2 weeks starting 1 week after simian immunodeficiency virus inoculation. Despite lower plasma TFV levels in the oral TDF arm, similar TFV diphosphate levels and antiviral activities were measured in lymphoid cells of most tissues. In intestinal tissues, however, oral TDF resulted in higher active drug levels, associated with lower virus levels and better immune preservation.
- Published
- 2012
- Full Text
- View/download PDF
50. Metabolic activation of the anti-hepatitis C virus nucleotide prodrug PSI-352938.
- Author
-
Niu C, Tolstykh T, Bao H, Park Y, Babusis D, Lam AM, Bansal S, Du J, Chang W, Reddy PG, Zhang HR, Woolley J, Wang LQ, Chao PB, Ray AS, Otto MJ, Sofia MJ, Furman PA, and Murakami E
- Subjects
- Cells, Cultured, Cytochrome P-450 CYP3A metabolism, Guanylate Kinases metabolism, Hepatocytes metabolism, Humans, Nucleoside-Diphosphate Kinase metabolism, Phosphoric Diester Hydrolases metabolism, Antiviral Agents metabolism, Cyclic P-Oxides metabolism, Hepacivirus drug effects, Nucleosides metabolism
- Abstract
PSI-352938 is a novel cyclic phosphate prodrug of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine-5'-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3',5'-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O(6)-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5'-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically.
- Published
- 2012
- Full Text
- View/download PDF
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