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Heo M, Norton BL, Pericot-Valverde I, Mehta SH, Tsui JI, Taylor LE, Lum PJ, Feinberg J, Kim AY, Arnsten JH, Sprecht-Walsh S, Page K, Murray-Krezan C, Anderson J, Litwin AH. Optimal hepatitis C treatment adherence patterns and sustained virologic response among people who inject drugs: The HERO study. J Hepatol 2024; 80:702-713. [PMID: 38242324 DOI: 10.1016/j.jhep.2023.12.020] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2023] [Revised: 11/27/2023] [Accepted: 12/20/2023] [Indexed: 01/21/2024]
Abstract
BACKGROUND & AIMS Direct-acting antivirals (DAAs) are highly effective for treating HCV infection even among people who inject drugs (PWID). Yet, little is known about patients' adherence patterns and their association with sustained virologic response (SVR) rates. We aimed to summarize various adherence patterns and determine their associations with SVR. METHODS Electronic blister packs were used to measure daily adherence to once-a-day sofosbuvir/velpatasvir during the 12-week treatment period among active PWIDs. Blister pack data were available for 496 participants who initiated DAAs for whom SVR status was known. Adherence was summarized in multiple patterns, such as total adherent days, consecutive missed days, and early discontinuations. Thresholds for adherence patterns associated with >90% SVR rates were also determined. RESULTS The overall SVR rate was 92.7%, with a median adherence rate of 75%. All adherence patterns indicating greater adherence were significantly associated with achieving SVR. Participant groups with ≥50% (>42/84) adherent days or <26 consecutive missed days achieved an SVR rate of >90%. Greater total adherent days during 9-12 weeks and no early discontinuation were significantly associated with higher SVR rates only in those with <50% adherence. Participants with first month discontinuation and ≥2 weeks of treatment interruption had low SVR rates, 25% and 85%, respectively. However, greater adherent days were significantly associated with SVR (adjusted odds ratio 1.10; 95% CI 1.04-1.16; p <0.001) even among participants with ≥14 consecutive missed days. CONCLUSIONS High SVR rates can be achieved in the PWID population despite suboptimal adherence. Encouraging patients to take as much medication as possible, with <2 weeks consecutive missed days and without early discontinuation, was found to be important for achieving SVR. IMPACT AND IMPLICATIONS People who inject drugs can be cured of HCV in >90% of cases, even with relatively low adherence to direct-acting antivirals, but early discontinuations and long treatment interruptions can significantly reduce the likelihood of achieving cure. Clinicians should encourage people who inject drugs who are living with HCV to adhere daily to direct-acting antivirals as consistently as possible, but if any days are interrupted, to continue and complete treatment. These results from the HERO study are important for patients living with HCV, clinicians, experts writing clinical guidelines, and payers. CLINICAL TRIAL NUMBER NCT02824640.
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Affiliation(s)
- Moonseong Heo
- Department of Public Health Sciences, Clemson University, Clemson, SC 29605, USA.
| | - Brianna L Norton
- Department of Medicine, Albert Einstein College of Medicine/Montefiore Medical Center, 3330 Kossuth Avenue Bronx, NY 10467, USA
| | - Irene Pericot-Valverde
- Department of Psychology, College of Behavioral, Social, and Health Sciences, Clemson University, Clemson, SC 29634, USA
| | - Shruti H Mehta
- Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E6546, Baltimore, MD 21205, USA
| | - Judith I Tsui
- Department of Medicine, University of Washington, 325 9th Ave., Seattle, WA 98104, USA
| | - Lynn E Taylor
- Department of Pharmacy, University of Rhode Island, Avedesian Hall, 7 Greenhouse Rd, Kingston, RI 02881, USA
| | - Paula J Lum
- Division of HIV, Infectious Disease and Global Medicine, University of California, San Francisco and San Francisco General Hospital, 2540 23rd Street, San Francisco, CA 94110, USA
| | - Judith Feinberg
- Department of Behavioral Medicine and Psychiatry, and Department of Medicine, Section of Infectious Diseases, West Virginia University School of Medicine, 930 Chestnut Ridge Road, Morgantown, WV 26505, USA
| | - Arthur Y Kim
- Division of Infectious Diseases, Massachusetts General Hospital and Harvard Medical School, 55 Fruit St., Boston, MA 02114, USA
| | - Julia H Arnsten
- Department of Medicine, Albert Einstein College of Medicine/Montefiore Medical Center, 3330 Kossuth Avenue Bronx, NY 10467, USA
| | | | - Kimberly Page
- Department of Internal Medicine, University of New Mexico Health Sciences Center, University of New Mexico MSC 10 5550, Albuquerque, NM 87131, USA
| | - Cristina Murray-Krezan
- Division of General Internal Medicine, Department of Medicine, University of Pittsburgh School of Medicine, 200 Meyran Avenue, Suite 300, Pittsburgh, PA 15213, USA
| | - Jessica Anderson
- Department of Internal Medicine, University of New Mexico Health Sciences Center, University of New Mexico MSC 10 5550, Albuquerque, NM 87131, USA
| | - Alain H Litwin
- School of Health Research, Clemson University, Clemson, SC 29605, USA; Department of Medicine, University of South Carolina School of Medicine, 876 W Faris Rd, Greenville, SC 29605, USA; Department of Medicine, Prisma Health, Greenville, SC 29605, USA.
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Reingardt DE, Ostankova YV, Lyalina LV, Anufrieva EV, Semenov AV, Totolian AA. Distribution of hepatitis С virus drug resistance mutations among patients with recurrence of the disease during therapy with direct antiviral drugs. HIV INFECTION AND IMMUNOSUPPRESSIVE DISORDERS 2024; 15:86-93. [DOI: 10.22328/2077-9828-2023-15-4-86-93] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
Abstract
The aim of the study was to identify the prevalence of drug resistance mutations in the hepatitis C virus among patients with relapse of the disease on therapy with direct antiviral drugs.Materials and methods. The study material included 31 blood plasma samples from patients with chronic hepatitis C with relapse of the disease on therapy with direct antiviral drugs. Samples were screened for the presence of HCV RNA. In case of detection of HCV RNA, amplification was carried out using a set of primers jointly flanking the NS3, NS5A, NS5B genes. After sequencing the nucleotide sequences of these genes, the subtype of the virus was determined and drug resistance mutations were identified.Results and discussion. The age of the patients ranged from 33 to 62 and averaged 45.8±8.38 years. The number of men in the group prevailed compared to women — 21 (67%) and 10 (33%), respectively. Viral load determination results ranged from 3.1×103 to 4.2×107 IU/ml. The distribution of genotypes was as follows: 1a — 26% (n=8), 1b — 29% (n=9), 3a — 45% (n=14). The nucleotide sequence of the NS3, NS5A, NS5B regions was determined in all samples. Mutations associated with drug resistance were detected in 87% (n=27). In all identified cases, the mutations resulted in viral resistance to at least one drug included in the patient’s current treatment regimen. In one patient, amino acid substitutions were found in three regions at once, which led to the emergence of resistance to two drugs in the regimen.Conclusion. Conducting a preliminary examination of patients to identify mutations of drug resistance to direct antiviral drugs can affect the effectiveness of the planned treatment and the choice of the optimal regimen.
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Affiliation(s)
| | | | | | | | - A. V. Semenov
- State Scientific Center of Virology and Biotechnology «Vector»
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Faiz S, Irfan M, Farooq S, Khan IA, Iqbal H, Wahab AT, Shakeel M, Gong P, Iftner T, Choudhary MI. Study of drug resistance-associated genetic mutations, and phylo-genetic analysis of HCV in the Province of Sindh, Pakistan. Sci Rep 2023; 13:12213. [PMID: 37500705 PMCID: PMC10374889 DOI: 10.1038/s41598-023-39339-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2023] [Accepted: 07/24/2023] [Indexed: 07/29/2023] Open
Abstract
Current management of HCV infection is based on Direct-Acting Antiviral Drugs (DAAs). However, resistance-associated mutations, especially in the NS3 and NS5B regions are gradually decreasing the efficacy of DAAs. The aim of the current study was to identify such mutations in the NS3, and NS5B genes in DAAs treatment-naïve Pakistani chronic HCV 3a patients. Peripheral blood samples were collected from 233 chronic HCV 3a patients at different tertiary care hospitals in Karachi, Pakistan, between August 2020 to September 2021. PCR-amplified target regions of the NS3/NS5B gene were subjected to Sanger sequencing to identify resistance-associated mutations. Phylogenetic analysis of the identified amino acid sequences was performed using HCV3a sequences of the global population in the virus pathogen resource (VIPR) database. Sequence analysis identified five amino acid mutations, Leu36Pro, Gln41His, Gln80Lys/Arg, Ala156Tyr, and Gln168Arg in the NS3 region, and two mutations Leu159Phe and Cys316Arg in the NS5B region. Phylogenetic analysis revealed a high genetic diversity in the studied isolates. Overall, the prevalence of resistance-associated substitutions was almost similar to other geographic regions worldwide. This data could be helpful in selecting the most effective treatment regimen for HCV chronically infected people in Pakistan.
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Affiliation(s)
- Sirmast Faiz
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, National Institute of Virology, University of Karachi, Karachi, 75270, Pakistan
| | - Muhammad Irfan
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, Jamil-ur-Rahman Center for Genome Research, University of Karachi, Karachi, 75270, Pakistan
| | - Saba Farooq
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, National Institute of Virology, University of Karachi, Karachi, 75270, Pakistan.
| | - Ishtiaq Ahmad Khan
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, Jamil-ur-Rahman Center for Genome Research, University of Karachi, Karachi, 75270, Pakistan.
| | - Hana'a Iqbal
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, National Institute of Virology, University of Karachi, Karachi, 75270, Pakistan
| | - Atia-Tul Wahab
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan
| | - Muhammad Shakeel
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, Jamil-ur-Rahman Center for Genome Research, University of Karachi, Karachi, 75270, Pakistan
| | - Peng Gong
- Wuhan Institute of Virology, Chinese Academy of Sciences, No.44 Xiao Hong Shan, Wuhan, 430071, Hubei, China
| | - Thomas Iftner
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, National Institute of Virology, University of Karachi, Karachi, 75270, Pakistan
- Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital and Medical Faculty, Eberhard Karls University, Tuebingen, Germany
| | - M Iqbal Choudhary
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, National Institute of Virology, University of Karachi, Karachi, 75270, Pakistan.
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, Jamil-ur-Rahman Center for Genome Research, University of Karachi, Karachi, 75270, Pakistan.
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan.
- Dr. Panjwani Centre for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, 75270, Pakistan.
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Kneller D, Phillips G, Weiss KL, Zhang Q, Coates L, Kovalevsky A. Direct Observation of Protonation State Modulation in SARS-CoV-2 Main Protease upon Inhibitor Binding with Neutron Crystallography. J Med Chem 2021; 64:4991-5000. [PMID: 33755450 PMCID: PMC8009097 DOI: 10.1021/acs.jmedchem.1c00058] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2021] [Indexed: 02/08/2023]
Abstract
The main protease (3CL Mpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. To date, no small-molecule clinical drugs are available that specifically inhibit SARS-CoV-2 Mpro. To aid rational drug design, we determined a neutron structure of Mpro in complex with the α-ketoamide inhibitor telaprevir at near-physiological (22 °C) temperature. We directly observed protonation states in the inhibitor complex and compared them with those in the ligand-free Mpro, revealing modulation of the active-site protonation states upon telaprevir binding. We suggest that binding of other α-ketoamide covalent inhibitors can lead to the same protonation state changes in the Mpro active site. Thus, by studying the protonation state changes induced by inhibitors, we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.
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Affiliation(s)
- Daniel
W. Kneller
- Neutron
Scattering Division, Oak Ridge National
Laboratory, 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States
- National
Virtual Biotechnology Laboratory, US Department of Energy, Washington, D.C. 20585, United States
| | - Gwyndalyn Phillips
- Neutron
Scattering Division, Oak Ridge National
Laboratory, 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States
- National
Virtual Biotechnology Laboratory, US Department of Energy, Washington, D.C. 20585, United States
| | - Kevin L. Weiss
- Neutron
Scattering Division, Oak Ridge National
Laboratory, 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States
- National
Virtual Biotechnology Laboratory, US Department of Energy, Washington, D.C. 20585, United States
| | - Qiu Zhang
- Neutron
Scattering Division, Oak Ridge National
Laboratory, 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States
- National
Virtual Biotechnology Laboratory, US Department of Energy, Washington, D.C. 20585, United States
| | - Leighton Coates
- National
Virtual Biotechnology Laboratory, US Department of Energy, Washington, D.C. 20585, United States
- Second
Target Station, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States
| | - Andrey Kovalevsky
- Neutron
Scattering Division, Oak Ridge National
Laboratory, 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States
- National
Virtual Biotechnology Laboratory, US Department of Energy, Washington, D.C. 20585, United States
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Heo M, Pericot-Valverde I, Rennert L, Akiyama MJ, Norton BL, Gormley M, Agyemang L, Arnsten JH, Litwin AH. Hepatitis C virus DAA treatment adherence patterns and SVR among people who inject drugs treated in opioid agonist therapy programs. Clin Infect Dis 2021; 73:2093-2100. [PMID: 33876230 DOI: 10.1093/cid/ciab334] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Indexed: 11/12/2022] Open
Abstract
BACKGROUND Adequate medication adherence is critical for achieving sustained viral response (SVR) of hepatitis C virus (HCV) among people who inject drugs (PWID). However, it is less known which patterns of direct-acting antiviral (DAA) treatment adherence are associated with SVR in this population or what factors are associated with each pattern. METHODS The randomized three-arm PREVAIL study utilized electronic blister packs to obtain daily time frame adherence data in opiate agonist therapy program settings. Exact logistic regressions were applied to test the associations between SVR and six types of treatment adherence patterns. RESULTS Of the 113 participants treated with combination DAAs, 109 (96.5%) achieved SVR. SVR was significantly associated with all pattern parameters except for number of switches between adherent and missed days: total adherent daily doses (exact AOR=1.12; 95%CI=1.04-1.22), percent total doses (1.09; 1.03-1.16), days on treatment (1.16; 1.05-1.32), maximum consecutive adherent days (1.34; 1.06-2.04), maximum consecutive non-adherent days (.85; .74-.95=.003). SVR was significantly associated with total adherent doses in the first two months of treatment, it was not in the last month. Compared to White participants (30.7±11.8(se)), Black (18.4±7.8) and Hispanic participants (19.2±6.1) had significantly shorter maximum consecutive adherent days. While alcohol intoxication was significantly associated with frequent switches, drug use was not associated with any adherence pattern. CONCLUSION Consistent maintenance of adequate total dose adherence over the entire course of HCV treatment is important in achieving SVR among PWID. Additional integrative addiction and medical care may be warranted for treating PWID experiencing alcohol intoxication.
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Affiliation(s)
- Moonseong Heo
- Department of Public Health Sciences, Clemson University, Clemson, SC, USA
| | | | - Lior Rennert
- Department of Public Health Sciences, Clemson University, Clemson, SC, USA
| | - Matthew J Akiyama
- Department of Medicine, Division of General Internal Medicine, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Brianna L Norton
- Department of Medicine, Division of General Internal Medicine, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Mirinda Gormley
- Department of Public Health Sciences, Clemson University, Clemson, SC, USA
| | - Linda Agyemang
- Department of Medicine, Division of General Internal Medicine, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Julia H Arnsten
- Department of Medicine, Division of General Internal Medicine, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Alain H Litwin
- Clemson University School of Health Research, Clemson University, Clemson, SC, USA.,Department of Medicine, University of South Carolina School of Medicine, Greenville, SC, USA Department of Internal Medicine, Prisma Health, Greenville, SC, USA
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Mohan S, Elhassan Taha MM, Makeen HA, Alhazmi HA, Al Bratty M, Sultana S, Ahsan W, Najmi A, Khalid A. Bioactive Natural Antivirals: An Updated Review of the Available Plants and Isolated Molecules. Molecules 2020; 25:E4878. [PMID: 33105694 PMCID: PMC7659943 DOI: 10.3390/molecules25214878] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Revised: 10/12/2020] [Accepted: 10/14/2020] [Indexed: 12/17/2022] Open
Abstract
Viral infections and associated diseases are responsible for a substantial number of mortality and public health problems around the world. Each year, infectious diseases kill 3.5 million people worldwide. The current pandemic caused by COVID-19 has become the greatest health hazard to people in their lifetime. There are many antiviral drugs and vaccines available against viruses, but they have many disadvantages, too. There are numerous side effects for conventional drugs, and active mutation also creates drug resistance against various viruses. This has led scientists to search herbs as a source for the discovery of more efficient new antivirals. According to the World Health Organization (WHO), 65% of the world population is in the practice of using plants and herbs as part of treatment modality. Additionally, plants have an advantage in drug discovery based on their long-term use by humans, and a reduced toxicity and abundance of bioactive compounds can be expected as a result. In this review, we have highlighted the important viruses, their drug targets, and their replication cycle. We provide in-depth and insightful information about the most favorable plant extracts and their derived phytochemicals against viral targets. Our major conclusion is that plant extracts and their isolated pure compounds are essential sources for the current viral infections and useful for future challenges.
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MESH Headings
- Antiviral Agents/chemistry
- Antiviral Agents/classification
- Antiviral Agents/isolation & purification
- Antiviral Agents/therapeutic use
- Betacoronavirus/drug effects
- Betacoronavirus/pathogenicity
- Betacoronavirus/physiology
- COVID-19
- Coronavirus Infections/drug therapy
- Coronavirus Infections/pathology
- Coronavirus Infections/virology
- Drug Discovery
- HIV/drug effects
- HIV/pathogenicity
- HIV/physiology
- HIV Infections/drug therapy
- HIV Infections/pathology
- HIV Infections/virology
- Hepacivirus/drug effects
- Hepacivirus/pathogenicity
- Hepacivirus/physiology
- Hepatitis C, Chronic/drug therapy
- Hepatitis C, Chronic/pathology
- Hepatitis C, Chronic/virology
- Herpes Simplex/drug therapy
- Herpes Simplex/pathology
- Herpes Simplex/virology
- Humans
- Influenza, Human/drug therapy
- Influenza, Human/pathology
- Influenza, Human/virology
- Orthomyxoviridae/drug effects
- Orthomyxoviridae/pathogenicity
- Orthomyxoviridae/physiology
- Pandemics
- Phytochemicals/chemistry
- Phytochemicals/classification
- Phytochemicals/isolation & purification
- Phytochemicals/therapeutic use
- Plants, Medicinal
- Pneumonia, Viral/drug therapy
- Pneumonia, Viral/pathology
- Pneumonia, Viral/virology
- SARS-CoV-2
- Simplexvirus/drug effects
- Simplexvirus/pathogenicity
- Simplexvirus/physiology
- Virus Internalization/drug effects
- Virus Replication/drug effects
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Affiliation(s)
- Syam Mohan
- Substance Abuse and Toxicology Research Centre, Jazan University, Jazan 45142, Saudi Arabia; (M.M.E.T.); (H.A.A.); (A.K.)
| | - Manal Mohamed Elhassan Taha
- Substance Abuse and Toxicology Research Centre, Jazan University, Jazan 45142, Saudi Arabia; (M.M.E.T.); (H.A.A.); (A.K.)
| | - Hafiz A. Makeen
- Department of Clinical Pharmacy, College of Pharmacy, Jazan University, Jazan 45142, Saudi Arabia;
| | - Hassan A. Alhazmi
- Substance Abuse and Toxicology Research Centre, Jazan University, Jazan 45142, Saudi Arabia; (M.M.E.T.); (H.A.A.); (A.K.)
- Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, Jazan 45142, Saudi Arabia; (M.A.B.); (W.A.); (A.N.)
| | - Mohammed Al Bratty
- Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, Jazan 45142, Saudi Arabia; (M.A.B.); (W.A.); (A.N.)
| | - Shahnaz Sultana
- Department of Pharmacognosy, College of Pharmacy, Jazan University, Jazan 45142, Saudi Arabia;
| | - Waquar Ahsan
- Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, Jazan 45142, Saudi Arabia; (M.A.B.); (W.A.); (A.N.)
| | - Asim Najmi
- Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, Jazan 45142, Saudi Arabia; (M.A.B.); (W.A.); (A.N.)
| | - Asaad Khalid
- Substance Abuse and Toxicology Research Centre, Jazan University, Jazan 45142, Saudi Arabia; (M.M.E.T.); (H.A.A.); (A.K.)
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7
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Farouk F, Wahba D, Mogawer S, Elkholy S, Elmeligui A, Abdelghani R, Ibahim S. Development and Validation of a New LC-MS/MS Analytical Method for Direct-Acting Antivirals and Its Application in End-Stage Renal Disease Patients. Eur J Drug Metab Pharmacokinet 2020; 45:89-99. [PMID: 31667795 DOI: 10.1007/s13318-019-00584-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND AND OBJECTIVE The effectiveness of direct-acting antivirals (DAAs) is not well established in end-stage renal disease (ESRD) patients. Assessment of the plasma concentrations may support understanding of their therapeutic outcomes in this population. The aim of this study is to develop a direct, yet matrix-effect tolerant, analytical method for determining DAAs in the plasma of ESRD patients while maintaining a moderate cost per sample and with an improved analyte extraction recovery. METHODS In this study, a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the analysis of ombitasvir (OMB), paritaprevir (PRT) and ritonavir (RIT) in plasma. Sample preparation was performed using the liquid-liquid extraction (LLE) method. Isocratic separation was performed using a mixture of methanol and 10 mM ammonium acetate (79:21, v/v) followed by MS/MS detection. The method was validated and applied to determine DAAs in the plasma of ESRD patients (n = 7). RESULTS The developed method was linear (r2 > 0.995), accurate (89.4 ± 7.8 to 108.3 ± 3.0) and precise (% CV 0.9-15.0) and showed improved recovery (> 80) over previously published ones in the range 5-250, 30-1,500, 20-1,000 ng/mL for OMB, PRT and RIT, respectively. Relative matrix effect was absent, and the method accurately determined the three DAAs in real-life samples (n = 7). CONCLUSIONS An efficient analytical method for the determination of DAAs is presented. The method overcomes the potential analytical response fluctuation in ESRD. The developed method show improved extraction recoveries and is suitable for routine application in developing economies where hepatitis C virus is most prevalent.
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Affiliation(s)
- Faten Farouk
- Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ahram Canadian University, 6th October City, Egypt.
| | - Dina Wahba
- National Organization of Drug Quality Control and Research, Giza, Egypt
| | - Sherif Mogawer
- Internal Medicine Department, Hepato-gastroenterology Unit, Faculty of Medicine, Cairo University, Giza, Egypt
| | - Shaimaa Elkholy
- Internal Medicine Department, Hepato-gastroenterology Unit, Faculty of Medicine, Cairo University, Giza, Egypt
| | - Ahmed Elmeligui
- Internal Medicine Department, Hepato-gastroenterology Unit, Faculty of Medicine, Cairo University, Giza, Egypt
| | - Reham Abdelghani
- Internal Medicine Department, Nephrology Unit, Faculty of Medicine, Cairo University, Giza, Egypt
| | - Salwa Ibahim
- Internal Medicine Department, Nephrology Unit, Faculty of Medicine, Cairo University, Giza, Egypt
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8
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Veselovská L, Kudlová N, Gurská S, Lišková B, Medvedíková M, Hodek O, Tloušťová E, Milisavljevic N, Tichý M, Perlíková P, Mertlíková‐Kaiserová H, Trylčová J, Pohl R, Klepetářová B, Džubák P, Hajdúch M, Hocek M. Synthesis and Cytotoxic and Antiviral Activity Profiling of All‐Four Isomeric Series of Pyrido‐Fused 7‐Deazapurine Ribonucleosides. Chemistry 2020; 26:13002-13015. [DOI: 10.1002/chem.202001124] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2020] [Indexed: 12/16/2022]
Affiliation(s)
- Lucia Veselovská
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
| | - Natálie Kudlová
- Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University and University Hospital in Olomouc Hněvotínská 5 775 15 Olomouc Czech Republic
- Cancer Research Czech Republic Hněvotínská 5 775 15 Olomouc Czech Republic
| | - Soňa Gurská
- Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University and University Hospital in Olomouc Hněvotínská 5 775 15 Olomouc Czech Republic
- Cancer Research Czech Republic Hněvotínská 5 775 15 Olomouc Czech Republic
| | - Barbora Lišková
- Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University and University Hospital in Olomouc Hněvotínská 5 775 15 Olomouc Czech Republic
| | - Martina Medvedíková
- Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University and University Hospital in Olomouc Hněvotínská 5 775 15 Olomouc Czech Republic
| | - Ondřej Hodek
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
| | - Eva Tloušťová
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
| | - Nemanja Milisavljevic
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
- Department of Organic Chemistry Faculty of Science Charles University in Prague Hlavova 8 12843 Prague 2 Czech Republic
| | - Michal Tichý
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
| | - Pavla Perlíková
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
| | - Helena Mertlíková‐Kaiserová
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
| | - Jana Trylčová
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
| | - Radek Pohl
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
| | - Blanka Klepetářová
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
| | - Petr Džubák
- Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University and University Hospital in Olomouc Hněvotínská 5 775 15 Olomouc Czech Republic
- Cancer Research Czech Republic Hněvotínská 5 775 15 Olomouc Czech Republic
| | - Marián Hajdúch
- Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University and University Hospital in Olomouc Hněvotínská 5 775 15 Olomouc Czech Republic
- Cancer Research Czech Republic Hněvotínská 5 775 15 Olomouc Czech Republic
| | - Michal Hocek
- Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam. 2 16610 Prague 6 Czech Republic
- Department of Organic Chemistry Faculty of Science Charles University in Prague Hlavova 8 12843 Prague 2 Czech Republic
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9
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Analysis of drug-resistance-associated mutations and genetic barriers in hepatitis C virus NS5B sequences in China. Arch Virol 2020; 165:2013-2020. [PMID: 32601956 DOI: 10.1007/s00705-020-04713-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2019] [Accepted: 05/22/2020] [Indexed: 12/16/2022]
Abstract
The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase that is required for viral genome replication and constitutes the most important target region for drugs being developed as direct-acting antivirals (DAAs) against HCV genotype 1. However, the extreme genetic variability leading to drug resistance mutations and genetic barriers has dramatically compromised the effectiveness of DAA therapy. The purpose of this study was to analyze the genetic variability of NS5B polymerase in HCV patients from different provinces of China to identify the impact of these resistance sites on genetic barriers. We analyzed 3489 NS5B sequences of HCV strains circulating in different regions of China, obtained from the GenBank database, 153 of which were from three cities in Sichuan Province (Yibin, Zigong and Zhangzhou). Sequence alignment was conducted using MEGA 6.0, the genetic information was translated into amino acids, and the percentage of polymorphic amino acid sites was calculated. The Vijver method was used to evaluate the occurrence of genetic barriers in HCV NS5B sequences. Blood samples were collected from 153 HCV patients from Sichuan for NS5B sequence analysis using real-time PCR and the Sanger method. Of the 17 antiviral drug resistance sites summarized from the published literature, nine were found in Chinese NS5B sequences, and C316Y was identified as the dominant mutation. Analysis of genetic barriers revealed that the probability of mutation to a drug-resistance-associated amino acid, in response to selective pressure from antiviral drugs was 100% at site 96 and 99.7% at site 282. Our study is the first to analyze the drug resistance sites and to evaluate genetic barriers in NS5B sequences that could affect the responsiveness of Chinese HCV patients to DAA therapy. The results provide a valuable basis for drug development and introduction of foreign-origin antiviral drugs in China that targeting the HCV NS5B region.
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10
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Rahman M, Janjua NZ, Shafiq TKI, Chowdhury EI, Sarker MS, Khan SI, Reza M, Faruque MO, Kabir A, Anis AH, Azim T. Hepatitis C virus treatment in people who inject drugs (PWID) in Bangladesh. THE INTERNATIONAL JOURNAL OF DRUG POLICY 2019; 74:69-75. [DOI: https:/doi.org/10.1016/j.drugpo.2019.09.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2023]
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11
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Rahman M, Janjua NZ, Shafiq TKI, Chowdhury EI, Sarker MS, Khan SI, Reza M, Faruque MO, Kabir A, Anis AH, Azim T. Hepatitis C virus treatment in people who inject drugs (PWID) in Bangladesh. THE INTERNATIONAL JOURNAL OF DRUG POLICY 2019; 74:69-75. [PMID: 31542689 DOI: 10.1016/j.drugpo.2019.09.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2019] [Revised: 09/03/2019] [Accepted: 09/05/2019] [Indexed: 12/13/2022]
Abstract
BACKGROUND Given the considerable social marginalization experienced by people who inject drugs (PWID), treatment of hepatitis C virus (HCV) in this population presents unique challenges. This study assessed the feasibility of treating HCV infection with direct-acting antiviral (DAA) medications among PWID receiving harm reduction services from a Drop-in-Center in Dhaka, Bangladesh. METHODS In this prospective study conducted between December 2016 and May 2018, 200 PWID with either recent injecting drug use (i.e., within the previous two months) or a history of injecting drug use and are currently receiving opioid substitution therapy were recruited. Blood was collected to conduct relevant laboratory tests. Eligible PWID who tested positive for HCV RNA (n = 55), were provided daily daclatasvir (60 mg) and sofosbuvir (400 mg) for 12 weeks after which adherence level, sustained virologic response (SVR), and reinfection were assessed. RESULTS At baseline, 40% (n = 79) of the 200 participants recruited to the study tested positive for antibodies to HCV and 34% (n = 68) had detectable HCV RNA in their blood. Of 55 eligible PWID who initiated treatment, 93% (n = 51) completed treatment while 87% (n = 48) were available for follow-up SVR assessment, all of whom achieved SVR. Thus, intent-to-treat SVR was 87% and the modified intent-to-treat SVR was 100% with one reinfection (4•2 cases per 100 person-years). Further, 75% (i.e., 41 out of the 55 participants) were at least 90% adherent to therapy. CONCLUSION Our findings strongly suggest that HCV treatment using sofosbuvir+daclatasvir for PWID enrolled in existing harm reduction programs in Bangladesh is feasible but may require additional interventions such as Opioid Substitution Therapy, intense follow up by outreach workers, and services and counselling provided by full time clinicians.
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Affiliation(s)
- Mustafizur Rahman
- Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh.
| | - Naveed Zafar Janjua
- British Columbia Centre for Disease Control, Vancouver, BC, Canada; School of Population and Public Health, University of British Columbia, Vancouver, BC Canada; CIHR Canadian HIV Trials Network, Vancouver, BC Canada
| | - Tanveer Khan Ibne Shafiq
- Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
| | - Ezazul Islam Chowdhury
- Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
| | - Md Safiullah Sarker
- Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
| | - Sharful Islam Khan
- Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
| | - Masud Reza
- Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh
| | | | | | - Aslam H Anis
- School of Population and Public Health, University of British Columbia, Vancouver, BC Canada; CIHR Canadian HIV Trials Network, Vancouver, BC Canada
| | - Tasnim Azim
- James P. Grant School of Public Health, BRAC University, Dhaka, Bangladesh
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12
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Alqahtani SA, Sulkowski MS. The Role of Interferon for the Treatment of Chronic Hepatitis C Virus Infection. TOPICS IN MEDICINAL CHEMISTRY 2019:97-113. [DOI: 10.1007/7355_2018_59] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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13
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Tokarenko A, Lišková B, Smoleń S, Táborská N, Tichý M, Gurská S, Perlíková P, Frydrych I, Tloušt'ová E, Znojek P, Mertlíková-Kaiserová H, Poštová Slavětínská L, Pohl R, Klepetářová B, Khalid NUA, Wenren Y, Laposa RR, Džubák P, Hajdúch M, Hocek M. Synthesis and Cytotoxic and Antiviral Profiling of Pyrrolo- and Furo-Fused 7-Deazapurine Ribonucleosides. J Med Chem 2018; 61:9347-9359. [PMID: 30281308 DOI: 10.1021/acs.jmedchem.8b01258] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Three series of isomeric pyrrolo- and furo-fused 7-deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2',3':4,5]pyrrolo[2,3- d]pyrimidine and furo[2',3':4,5]pyrrolo[2,3- d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3',2',4,5]pyrrolo[2,3- d]pyrimidine nucleosides were inactive. The most active were the methyl, methoxy, and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.
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Affiliation(s)
- Anna Tokarenko
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic.,Department of Organic Chemistry, Faculty of Science , Charles University in Prague , Hlavova 8 , CZ-12843 Prague 2 , Czech Republic
| | - Barbora Lišková
- Faculty of Medicine and Dentistry, Institute of Molecular and Translational Medicine , Palacky University and University Hospital in Olomouc , Hněvotínská 5 , CZ-775 15 Olomouc , Czech Republic
| | - Sabina Smoleń
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic
| | - Natálie Táborská
- Faculty of Medicine and Dentistry, Institute of Molecular and Translational Medicine , Palacky University and University Hospital in Olomouc , Hněvotínská 5 , CZ-775 15 Olomouc , Czech Republic
| | - Michal Tichý
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic
| | - Soňa Gurská
- Faculty of Medicine and Dentistry, Institute of Molecular and Translational Medicine , Palacky University and University Hospital in Olomouc , Hněvotínská 5 , CZ-775 15 Olomouc , Czech Republic
| | - Pavla Perlíková
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic
| | - Ivo Frydrych
- Faculty of Medicine and Dentistry, Institute of Molecular and Translational Medicine , Palacky University and University Hospital in Olomouc , Hněvotínská 5 , CZ-775 15 Olomouc , Czech Republic
| | - Eva Tloušt'ová
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic
| | - Pawel Znojek
- Faculty of Medicine and Dentistry, Institute of Molecular and Translational Medicine , Palacky University and University Hospital in Olomouc , Hněvotínská 5 , CZ-775 15 Olomouc , Czech Republic
| | - Helena Mertlíková-Kaiserová
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic
| | - Lenka Poštová Slavětínská
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic
| | - Radek Pohl
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic
| | - Blanka Klepetářová
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic
| | - Noor-Ul-Ain Khalid
- Department of Pharmacology and Toxicology , University of Toronto , 1 King's College Circle, Room 4213 , Toronto , Ontario M5S 1A8 , Canada
| | - Yiqian Wenren
- Department of Pharmacology and Toxicology , University of Toronto , 1 King's College Circle, Room 4213 , Toronto , Ontario M5S 1A8 , Canada
| | - Rebecca R Laposa
- Department of Pharmacology and Toxicology , University of Toronto , 1 King's College Circle, Room 4213 , Toronto , Ontario M5S 1A8 , Canada
| | - Petr Džubák
- Faculty of Medicine and Dentistry, Institute of Molecular and Translational Medicine , Palacky University and University Hospital in Olomouc , Hněvotínská 5 , CZ-775 15 Olomouc , Czech Republic.,Cancer Research Czech Republic , Hněvotínská 5 , CZ-775 15 Olomouc , Czech Republic
| | - Marián Hajdúch
- Faculty of Medicine and Dentistry, Institute of Molecular and Translational Medicine , Palacky University and University Hospital in Olomouc , Hněvotínská 5 , CZ-775 15 Olomouc , Czech Republic.,Cancer Research Czech Republic , Hněvotínská 5 , CZ-775 15 Olomouc , Czech Republic
| | - Michal Hocek
- Institute of Organic Chemistry and Biochemistry , Czech Academy of Sciences , Flemingovo nam. 2 , CZ-16610 Prague 6 , Czech Republic.,Department of Organic Chemistry, Faculty of Science , Charles University in Prague , Hlavova 8 , CZ-12843 Prague 2 , Czech Republic
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14
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Ianevski A, Zusinaite E, Kuivanen S, Strand M, Lysvand H, Teppor M, Kakkola L, Paavilainen H, Laajala M, Kallio-Kokko H, Valkonen M, Kantele A, Telling K, Lutsar I, Letjuka P, Metelitsa N, Oksenych V, Bjørås M, Nordbø SA, Dumpis U, Vitkauskiene A, Öhrmalm C, Bondeson K, Bergqvist A, Aittokallio T, Cox RJ, Evander M, Hukkanen V, Marjomaki V, Julkunen I, Vapalahti O, Tenson T, Merits A, Kainov D. Novel activities of safe-in-human broad-spectrum antiviral agents. Antiviral Res 2018; 154:174-182. [PMID: 29698664 PMCID: PMC7113852 DOI: 10.1016/j.antiviral.2018.04.016] [Citation(s) in RCA: 51] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2018] [Revised: 04/16/2018] [Accepted: 04/20/2018] [Indexed: 12/03/2022]
Abstract
According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley fever virus. Thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases.
339 approved, investigational and experimental safe-in-human antivirals were identified. 59 compounds, which target ≥3 viral diseases, were selected. 55 of the 59 compounds were tested against 8 RNA and DNA viruses. 7 compounds were found to possess novel antiviral activities.
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Affiliation(s)
- Aleksandr Ianevski
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim 7028, Norway.
| | - Eva Zusinaite
- Institute of Technology, University of Tartu, Tartu 50090, Estonia.
| | - Suvi Kuivanen
- Department of Virology, University of Helsinki, Helsinki 00014, Finland.
| | - Mårten Strand
- Department of Clinical Microbiology, Umeå University, Umeå 90185, Sweden.
| | - Hilde Lysvand
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim 7491, Norway.
| | - Mona Teppor
- Institute of Technology, University of Tartu, Tartu 50090, Estonia.
| | - Laura Kakkola
- Institute of Biomedicine, University of Turku, Turku 20520, Finland.
| | | | - Mira Laajala
- Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä 40500, Finland.
| | - Hannimari Kallio-Kokko
- Department of Virology and Immunology, University of Helsinki, Helsinki University Hospital, Helsinki 00014, Finland.
| | - Miia Valkonen
- Helsinki University Hospital, Helsinki 00014, Finland.
| | - Anu Kantele
- Helsinki University Hospital, Helsinki 00014, Finland.
| | - Kaidi Telling
- Institute of Medical Microbiology, University of Tartu, Tartu 50411, Estonia.
| | - Irja Lutsar
- Institute of Medical Microbiology, University of Tartu, Tartu 50411, Estonia.
| | | | | | - Valentyn Oksenych
- St. Olavs Hospital, Trondheim University Hospital, Clinic of Medicine, Trondheim 7006, Norway.
| | - Magnar Bjørås
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim 7491, Norway.
| | - Svein Arne Nordbø
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim 7491, Norway; Department of Medical Microbiology, St. Olavs Hospital, Trondheim University Hospital, Trondheim 7006, Norway.
| | - Uga Dumpis
- Pauls Stradins Clinical University Hospital, Riga 1002, Latvia.
| | - Astra Vitkauskiene
- Department of Laboratory Medicine, Lithuanian University of Health Science, Kaunas 44307, Lithuania.
| | - Christina Öhrmalm
- Department of Medical Sciences, Uppsala University, Uppsala 75309, Sweden.
| | - Kåre Bondeson
- Department of Medical Sciences, Uppsala University, Uppsala 75309, Sweden.
| | - Anders Bergqvist
- Department of Medical Sciences, Uppsala University, Uppsala 75309, Sweden.
| | - Tero Aittokallio
- Institute for Molecular Medicine Finland, FIMM, University of Helsinki, Helsinki 00290, Finland; Department of Mathematics and Statistics, University of Turku, Turku 20014, Finland.
| | - Rebecca J Cox
- Influenza Centre, Department of Clinical Science, University of Bergen, Bergen 5021, Norway.
| | - Magnus Evander
- Department of Clinical Microbiology, Umeå University, Umeå 90185, Sweden.
| | - Veijo Hukkanen
- Institute of Biomedicine, University of Turku, Turku 20520, Finland.
| | - Varpu Marjomaki
- Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä 40500, Finland.
| | - Ilkka Julkunen
- Institute of Biomedicine, University of Turku, Turku 20520, Finland.
| | - Olli Vapalahti
- Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki 00014, Finland; Department of Veterinary Biosciences, University of Helsinki, Helsinki 00014, Finland.
| | - Tanel Tenson
- Institute of Technology, University of Tartu, Tartu 50090, Estonia.
| | - Andres Merits
- Institute of Technology, University of Tartu, Tartu 50090, Estonia.
| | - Denis Kainov
- Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim 7028, Norway; Institute of Technology, University of Tartu, Tartu 50090, Estonia.
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15
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Sun D, Dai M, Shen S, Li C, Yan X. Analysis of Naturally Occurring Resistance-Associated Variants to NS3/4A Protein Inhibitors, NS5A Protein Inhibitors, and NS5B Polymerase Inhibitors in Patients With Chronic Hepatitis C. Gene Expr 2018; 18:63-69. [PMID: 29221500 PMCID: PMC5885147 DOI: 10.3727/105221617x15100607143377] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The first NS3/4A hepatitis C virus (HCV) protease inhibitors telaprevir and boceprevir were approved in 2011, and both NS5A and NS5B polymerase inhibitors were launched. Recently, direct-acting antivirals (DAAs) have had a major impact on patients infected with HCV. HCV DAAs are highly effective antivirals with fewer side effects. DAAs have been developed for the treatment of HCV infection in combination with PEG-IFN-α/RBV as well as in IFN-free regimens. However, some drug resistance mutations occur when a single oral DAA is used for treatment, which indicates that there is a low-frequency drug resistance mutation in HCV patients before the application of antiviral drugs. Our research showed that natural resistance to HCV DAAs was found in treatment-naive CHC patients and that the drug resistance mutation rates differ in various HCV genotypes. Many challenges posed by natural resistance should be considered in the context of DAA therapies.
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Affiliation(s)
- Danhui Sun
- Department of Infectious Diseases, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, P.R. China
| | - Mingjia Dai
- Department of Infectious Diseases, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, P.R. China
| | - Shanshan Shen
- Department of Infectious Diseases, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, P.R. China
| | - Chunyang Li
- Department of Infectious Diseases, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, P.R. China
| | - Xuebing Yan
- Department of Infectious Diseases, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, P.R. China
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16
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Li Z, Liu Y, Zhang Y, Shao X, Luo Q, Guo X, Lin G, Cai Q, Zhao Z, Chong Y. Naturally Occurring Resistance-Associated Variants to Hepatitis C Virus Direct-Acting Antiviral Agents in Treatment-Naive HCV Genotype 6a-Infected Patients. BIOMED RESEARCH INTERNATIONAL 2017; 2017:9849823. [PMID: 29164151 PMCID: PMC5661091 DOI: 10.1155/2017/9849823] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 04/05/2017] [Revised: 05/31/2017] [Accepted: 07/10/2017] [Indexed: 12/18/2022]
Abstract
BACKGROUND AND OBJECTIVE The direct-acting antiviral agents (DAAs) antiviral therapy has drastically improved the prognosis of hepatitis C virus (HCV) patients. However, the viral drug resistance-associated variants (RAVs) can limit the efficacy of DAAs. For the HCV-6a is not the predominant prevalent genotype; the data on the prevalence of naturally occurring RAVs in it is scarce. Our study aims to assess the prevalence of RAVs in treatment-naive HCV-6a patients. METHODS Nested PCR assays were performed on 95 HCV-6a patients to amplify HCV viral regions of NS3, NS5A, and NS5B. RESULTS In NS3/4A region, we detected Q80K in 95.5% isolates (84/88) and D168E in 2.3% isolates (2/88). In NS5A region, we detected Q30R in 93.2% isolates (82/88), L31M in 4.6% isolates (4/88), and H58P in 6.8% isolates (6/88). In NS5B region, we detected A15G in 2.3% isolates (2/88), S96T in 1.1% isolates (1/88), and S282T in 20.7% isolates (17/88) and we detected I482L in 100% isolates (4/4), V494A in 50% isolates (2/4), and V499A in 100% isolates (4/4). CONCLUSIONS RAVs to DAAs preexist in treatment-naive HCV-6a patients. Further studies should address the issue of the impact of RAVs in response to DAA therapies for HCV-6a patients.
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Affiliation(s)
- Zhanyi Li
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
- Guangdong Provincial Key Laboratory of Liver Disease, Guangdong, China
| | - Ying Liu
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
- Guangdong Provincial Key Laboratory of Liver Disease, Guangdong, China
| | - Ying Zhang
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Xiaoqiong Shao
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Qiumin Luo
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Xiaoyan Guo
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Guoli Lin
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Qingxian Cai
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
- Guangdong Provincial Key Laboratory of Liver Disease, Guangdong, China
| | - Zhixin Zhao
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Yutian Chong
- Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China
- Guangdong Provincial Key Laboratory of Liver Disease, Guangdong, China
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17
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Sacchi A, Tumino N, Turchi F, Refolo G, Fimia G, Ciccosanti F, Montalbano M, Lionetti R, Taibi C, D'Offizi G, Casetti R, Bordoni V, Cimini E, Martini F, Agrati C. Dendritic cells activation is associated with sustained virological response to telaprevir treatment of HCV-infected patients. Clin Immunol 2017; 183:82-90. [PMID: 28736275 DOI: 10.1016/j.clim.2017.07.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2016] [Revised: 06/28/2017] [Accepted: 07/19/2017] [Indexed: 11/16/2022]
Abstract
First anti-HCV treatments, that include protease inhibitors in conjunction with IFN-α and Ribavirin, increase the sustained virological response (SVR) up to 80% in patients infected with HCV genotype 1. The effects of triple therapies on dendritic cell (DC) compartment have not been investigated. In this study we evaluated the effect of telaprevir-based triple therapy on DC phenotype and function, and their possible association with treatment outcome. HCV+ patients eligible for telaprevir-based therapy were enrolled, and circulating DC frequency, phenotype, and function were evaluated by flow-cytometry. The antiviral activity of plasmacytoid DC was also tested. In SVR patients, myeloid DC frequency transiently decreased, and returned to baseline level when telaprevir was stopped. Moreover, an up-regulation of CD80 and CD86 on mDC was observed in SVR patients as well as an improvement of IFN-α production by plasmacytoid DC, able to inhibit in vitro HCV replication.
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Affiliation(s)
- Alessandra Sacchi
- Cellular Immunology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy.
| | - Nicola Tumino
- Cellular Immunology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Federica Turchi
- Cellular Immunology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Giulia Refolo
- Cellular Biology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - GianMaria Fimia
- Cellular Biology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Fabiola Ciccosanti
- Cellular Biology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Marzia Montalbano
- Clinical Division, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Raffaella Lionetti
- Clinical Division, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Chiara Taibi
- Clinical Division, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Gianpiero D'Offizi
- Clinical Division, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Rita Casetti
- Cellular Immunology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Veronica Bordoni
- Cellular Immunology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Eleonora Cimini
- Cellular Immunology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Federico Martini
- Cellular Immunology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Chiara Agrati
- Cellular Immunology Laboratory, "Lazzaro Spallanzani" National Institute for Infectious Diseases, IRCCS, Rome, Italy
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18
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Li Z, Zhang Y, Liu Y, Shao X, Luo Q, Cai Q, Zhao Z. Naturally occurring drug resistance associated variants to hepatitis C virus direct-acting antiviral agents in treatment-naive HCV genotype 1b-infected patients in China. Medicine (Baltimore) 2017; 96:e6830. [PMID: 28489763 PMCID: PMC5428597 DOI: 10.1097/md.0000000000006830] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The direct-acting antiviral agents (DAAs) have drastically improved the prognosis of hepatitis C virus (HCV) patients. However, the resistance-associated variants (RAVs) to DAAs may hamper treatment. There was a lack of data on the prevalence of pre-exist RAVs in Chinese HCV-infected patients. We performed nested PCR assays on 74 HCV genotype 1b-infected patients to amplify HCV viral regions of NS3, NS5A, and NS5B to investigate the prevalence of RAVs to DAAs in treatment-naive HCV genotype1b-infected patients in China. The mutations A156S, T54S, and D168Y of the NS3/4A region were found in 18.33% (11/60), 6.67% (4/60), and 1.67% (1/60) of the successfully amplified cases. Mutations Q30R, L31M, and H58P of the NS5A region were confirmed in 57.63% (34/59), 1.69%(1/59), and 86.44% (51/59) of the cases. Mutations C316N, S365A, M414L, M423I, Y448H, I482T, I482 V, V494L, P495S, and V499A of the NS5B region were detected in 100% (60/60), 3.33% (2/60), 5.88% (3/51), 1.96% (1/51), 1.96% (1/51), 5.88% (3/51), 1.96% (1/51), 3.92% (2/51), 5.88% (3/51), and 15.69% (8/51) of cases, respectively. Naturally occurring RAVs to DAAs pre-exist in treatment-naive Chinese HCV genotype 1b-infected patients and the characteristic is different from that in Europe and the United States. Clinicians should consider RAVs upon the introduction of DAA-based antiviral therapy.
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Affiliation(s)
- Zhanyi Li
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou, Guangdong, China
| | - Ying Zhang
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou, Guangdong, China
| | - Ying Liu
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou, Guangdong, China
| | - Xiaoqiong Shao
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou, Guangdong, China
| | - QiuMin Luo
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou, Guangdong, China
| | - Qingxian Cai
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou, Guangdong, China
| | - Zhixin Zhao
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou, Guangdong, China
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Ikram A, Obaid A, Awan FM, Hanif R, Naz A, Paracha RZ, Ali A, Janjua HA. Identification of drug resistance and immune-driven variations in hepatitis C virus (HCV) NS3/4A, NS5A and NS5B regions reveals a new approach toward personalized medicine. Antiviral Res 2017; 137:112-124. [PMID: 27984060 DOI: 10.1016/j.antiviral.2016.10.013] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2016] [Revised: 10/06/2016] [Accepted: 10/21/2016] [Indexed: 02/07/2023]
Abstract
Cellular immune responses (T cell responses) during hepatitis C virus (HCV) infection are significant factors for determining the outcome of infection. HCV adapts to host immune responses by inducing mutations in its genome at specific sites that are important for HLA processing/presentation. Moreover, HCV also adapts to resist potential drugs that are used to restrict its replication, such as direct-acting antivirals (DAAs). Although DAAs have significantly reduced disease burden, resistance to these drugs is still a challenge for the treatment of HCV infection. Recently, drug resistance mutations (DRMs) observed in HCV proteins (NS3/4A, NS5A and NS5B) have heightened concern that the emergence of drug resistance may compromise the effectiveness of DAAs. Therefore, the NS3/4A, NS5A and NS5B drug resistance variations were investigated in this study, and their prevalence was examined in a large number of protein sequences from all HCV genotypes. Furthermore, potential CD4+ and CD8+ T cell epitopes were predicted and their overlap with genetic variations was explored. The findings revealed that many reported DRMs within NS3/4A, NS5A and NS5B are not drug-induced; rather, they are already present in HCV strains, as they were also detected in HCV-naïve patients. This study highlights several hot spots in which HLA and drug selective pressure overlap. Interestingly, these overlapping mutations were frequently observed among many HCV genotypes. This study implicates that knowledge of the host HLA type and HCV subtype/genotype can provide important information in defining personalized therapy.
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Affiliation(s)
- Aqsa Ikram
- Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Pakistan
| | - Ayesha Obaid
- Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Pakistan
| | - Faryal Mehwish Awan
- Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Pakistan
| | - Rumeza Hanif
- Department of Healtcare Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Pakistan
| | - Anam Naz
- Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Pakistan
| | - Rehan Zafar Paracha
- Department of Computer Sciences, RCMS, National University of Sciences and Technology (NUST), Pakistan
| | - Amjad Ali
- Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Pakistan
| | - Hussnain Ahmed Janjua
- Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Pakistan.
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Minosse C, Giombini E, Bartolini B, Capobianchi MR, Garbuglia AR. Ultra-Deep Sequencing Characterization of HCV Samples with Equivocal Typing Results Determined with a Commercial Assay. Int J Mol Sci 2016; 17:E1679. [PMID: 27739414 PMCID: PMC5085712 DOI: 10.3390/ijms17101679] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2016] [Revised: 09/13/2016] [Accepted: 09/23/2016] [Indexed: 01/18/2023] Open
Abstract
Hepatitis C virus (HCV) is classified into seven phylogenetically distinct genotypes, which are further subdivided into related subtypes. Accurate assignment of genotype/subtype is mandatory in the era of directly acting antivirals. Several molecular methods are available for HCV genotyping; however, a relevant number of samples with indeterminate, mixed, or unspecified subtype results, or even with misclassified genotypes, may occur. Using NS5B direct (DS) and ultra-deep pyrosequencing (UDPS), we have tested 43 samples, which resulted in genotype 1 unsubtyped (n = 17), mixed infection (n = 17), or indeterminate (n = 9) with the Abbott RealTime HCV Genotype II assay. Genotype 1 was confirmed in 14/17 samples (82%): eight resulted in subtype 1b, and five resulted in subtype 1a with both DS and UDPS, while one was classified as subtype 1e by DS and mixed infection (1e + 1a) by UDPS. Three of seventeen genotype 1 samples resulted in genotype 3h with both sequencing approaches. Only one mixed infection was confirmed by UDPS (4d + 1a), while in 88% of cases a single component of the mixture was detected (five genotype 1a, four genotype 1b, two genotype 3a, two genotype 4m, and two genotype 4d); 44% of indeterminate samples resulted genotype 2c by both DS and UDPS, 22% resulted genotype 3a; one indeterminate sample by Abbott resulted in genotype 4d, one resulted in genotype 6n, and one was classified as subtype 3a by DS, and resulted mixed infection (3a + 3h) by UDPS. The concordance between DS and UDPS was 94%, 88%, and 89% for genotype 1, co-infection, and indeterminate results, respectively. UDPS should be considered very useful to resolve ambiguous HCV genotyping results.
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Affiliation(s)
- Claudia Minosse
- Laboratory of Virology, National Institute for Infectious Diseases "Lazzaro Spallanzani"-IRCCS, Via Portuense 292, Rome 00149, Italy.
| | - Emanuela Giombini
- Laboratory of Virology, National Institute for Infectious Diseases "Lazzaro Spallanzani"-IRCCS, Via Portuense 292, Rome 00149, Italy.
| | - Barbara Bartolini
- Laboratory of Virology, National Institute for Infectious Diseases "Lazzaro Spallanzani"-IRCCS, Via Portuense 292, Rome 00149, Italy.
| | - Maria R Capobianchi
- Laboratory of Virology, National Institute for Infectious Diseases "Lazzaro Spallanzani"-IRCCS, Via Portuense 292, Rome 00149, Italy.
| | - Anna R Garbuglia
- Laboratory of Virology, National Institute for Infectious Diseases "Lazzaro Spallanzani"-IRCCS, Via Portuense 292, Rome 00149, Italy.
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Garbuglia AR, Visco-Comandini U, Lionetti R, Lapa D, Castiglione F, D’Offizi G, Taibi C, Montalbano M, Capobianchi MR, Paci P. Ultrasensitive HCV RNA Quantification in Antiviral Triple Therapy: New Insight on Viral Clearance Dynamics and Treatment Outcome Predictors. PLoS One 2016; 11:e0158989. [PMID: 27560794 PMCID: PMC4999094 DOI: 10.1371/journal.pone.0158989] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2016] [Accepted: 06/25/2016] [Indexed: 02/06/2023] Open
Abstract
OBJECTIVES Identifying the predictive factors of Sustained Virological Response (SVR) represents an important challenge in new interferon-based DAA therapies. Here, we analyzed the kinetics of antiviral response associated with a triple drug regimen, and the association between negative residual viral load at different time points during treatment. METHODS Twenty-three HCV genotype 1 (GT 1a n = 11; GT1b n = 12) infected patients were included in the study. Linear Discriminant Analysis (LDA) was used to establish possible association between HCV RNA values at days 1 and 4 from start of therapy and SVR. Principal component analysis (PCA) was applied to analyze the correlation between HCV RNA slope and SVR. A ultrasensitive (US) method was established to measure the residual HCV viral load in those samples which resulted "detected <12IU/ml" or undetectable with ABBOTT standard assay, and was retrospectively used on samples collected at different time points to establish its predictive power for SVR. RESULTS According to LDA, there was no association between SVR and viral kinetics neither at time points earlier than 1 week (days 1 and 4) after therapy initiation nor later. The slopes were not relevant for classifying patients as SVR or no-SVR. No significant differences were observed in the median HCV RNA values at T0 among SVR and no-SVR patients. HCV RNA values with US protocol (LOD 1.2 IU/ml) after 1 month of therapy were considered; the area under the ROC curve was 0.70. Overall, PPV and NPV of undetectable HCV RNA with the US method for SVR was 100% and 46.7%, respectively; sensitivity and specificity were 38.4% and 100% respectively. CONCLUSION HCV RNA "not detected" by the US method after 1 month of treatment is predictive of SVR in first generation Protease inhibitor (PI)-based triple therapy. The US method could have clinical utility for advanced monitoring of virological response in new interferon based DAA combination regimens.
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Affiliation(s)
- Anna Rosa Garbuglia
- Laboratory of Virology, “Lazzaro Spallanzani” National Institute for Infectious Diseases, IRCCS, Rome, Italy
- * E-mail:
| | - Ubaldo Visco-Comandini
- Clinical Department, “Lazzaro Spallanzani” National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Raffaella Lionetti
- Clinical Department, “Lazzaro Spallanzani” National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Daniele Lapa
- Laboratory of Virology, “Lazzaro Spallanzani” National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | | | - Gianpiero D’Offizi
- Clinical Department, “Lazzaro Spallanzani” National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Chiara Taibi
- Clinical Department, “Lazzaro Spallanzani” National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Marzia Montalbano
- Clinical Department, “Lazzaro Spallanzani” National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Maria Rosaria Capobianchi
- Laboratory of Virology, “Lazzaro Spallanzani” National Institute for Infectious Diseases, IRCCS, Rome, Italy
| | - Paola Paci
- Istituto di Analisi dei Sistemi ed Informatica “Antonio Ruberti” (IASI)—CNR, Rome, Italy
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Kliemann DA, Tovo CV, Gorini da Veiga AB, Machado AL, West J. Genetic Barrier to Direct Acting Antivirals in HCV Sequences Deposited in the European Databank. PLoS One 2016; 11:e0159924. [PMID: 27504952 PMCID: PMC4978475 DOI: 10.1371/journal.pone.0159924] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2016] [Accepted: 07/11/2016] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND & AIMS Development of resistance results from mutations in the viral genome, and the presence of selective drug pressure leads to the emergence of a resistant virus population. The aim of this study was to analyze the impact of genetic variability on the genetic barrier to drug resistance to DAAs. METHODS The genetic barrier was quantified based on the number and type of nucleotide mutations required to impart resistance, considering full-length HCV NS3, NS5A and NS5B regions segregated by genotype into subtypes 1a, 1b, 2a, 2b and 3a. This study analyzeds 789 NS3 sequences, 708 sequences and 536 NS5B sequences deposited in the European Hepatitis C Virus Database, in the following resistance-associated positions: NS3: F43/I/L/S/V, Q80K/R, R155K/G, A156G/S/T and D168A/C/E/G/H/N/T/V/Y; NS5A: L/M28A/T/V, Q30E/H/R, L31F/I/M/V, H58D or P58S and Y93C/F/H/N/S; NS5B: S282P/R/T, C316H/N/Y, S368T, Y448C/H, S556G/R, D559R. RESULTS Variants that require only one transversion in NS3 were found in 4 positions and include F43S, R80K, R155K/G and A156T. The genetic barrier to resistance shows subtypic differences at position 155 of the NS3 gene where a single transition is necessary in subtype 1a. In the NS5A gene, 5 positions where only one nucleotide change can confer resistance were found, such as L31M which requires one transversion in all subtypes, except in 0.28% of 1b sequences; and R30H, generated by a single transition, which was found in 10.25% of the sequences of genotype 1b. Other subtypic differences were observed at position 58, where resistance is less likely in genotype 1a because a transversion is required to create the variant 58S. For the NS5B inhibitors, the genetic barrier at positions conferring resistance was nearly identical in subtypes 1a and 1b, and single transitions or transversions were necessary in 5 positions to generate a drug-resistant variant of HCV. The positions C316Y and S556D required only one transition in all genotypes, Y448H and S556 G/N/R positions required only one transition for up to 98.8% of the sequences analyzed. A single variant in position 448 in genotype 1a is less likely to become the resistance variant 448H because it requires two transversions. Also, in the position 559D a transversion and a transition were necessary to generate the resistance mutant D559H. CONCLUSION Results revealed that in 14 out of 16 positions, conversion to a drug-resistant variant of HCV required only one single nucleotide substitutions threatening direct acting antivirals from all three classes.
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Affiliation(s)
- Dimas Alexandre Kliemann
- Graduate Program in Medicine, Hepatology, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil
- Medical Infectologist, Hospital Nossa Senhora da Conceição (HNSC), Porto Alegre, RS, Brazil
| | - Cristiane Valle Tovo
- Graduate Program in Medicine, Hepatology, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil
| | - Ana Beatriz Gorini da Veiga
- Graduate Program in Medicine, Hepatology, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil
- Department of Basic Health Sciences, Laboratory of Molecular Biology, Graduate Program in Pathology, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Brazil
| | - André Luiz Machado
- Graduate Program in Medicine, Hepatology, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil
- Medical Infectologist, Hospital Nossa Senhora da Conceição (HNSC), Porto Alegre, RS, Brazil
| | - John West
- School of Biological Sciences, Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska, United States of America, University of Nebraska, Lincoln, NE, United States of America
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23
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Cuypers L, Ceccherini-Silberstein F, Van Laethem K, Li G, Vandamme AM, Rockstroh JK. Impact of HCV genotype on treatment regimens and drug resistance: a snapshot in time. Rev Med Virol 2016; 26:408-434. [PMID: 27401933 DOI: 10.1002/rmv.1895] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2016] [Revised: 06/11/2016] [Accepted: 06/15/2016] [Indexed: 12/11/2022]
Abstract
The introduction of highly potent direct-acting antivirals (DAAs) has revolutionized hepatitis C virus treatment. Nevertheless, viral eradication worldwide remains a challenge also in the era of DAA treatment, because of the high associated costs, high numbers of undiagnosed patients, high re-infection rates in some risk groups and suboptimal drug efficacies associated with host and viral factors as well as advanced stages of liver disease. A correct determination of the HCV genotype allows administration of the most appropriate antiviral regimen. Additionally, HCV genetic sequencing improves our understanding of resistance-associated variants, either naturally occurring before treatment, acquired by transmission at HCV infection, or emerging after virological failure. Because treatment response rates, and the prevalence and development of drug resistance variants differ for each DAA regimen and HCV genotype, this review summarizes treatment opportunities per HCV genotype, and focuses on viral genetic sequencing to guide clinical decision making. Although approval of the first pan-genotypic DAA-only regimen is expected soon, HCV genetic sequencing will remain important because when DAA therapies fail, genotyping and resistance testing to select a new active DAA combination will be essential. Copyright © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Lize Cuypers
- KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Leuven, Belgium
| | | | - Kristel Van Laethem
- KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Leuven, Belgium
| | - Guangdi Li
- KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Leuven, Belgium.,Department of Metabolism and Endocrinology, Metabolic Syndrome Research Center, Key Laboratory of Diabetes Immunology, Ministry of Education, National Clinical Research Center for Metabolic Diseases, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Anne-Mieke Vandamme
- KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Leuven, Belgium.,Center for Global Health and Tropical Medicine, Microbiology Unit, Institute for Hygiene and Tropical Medicine, University Nova de Lisboa, Lisbon, Portugal
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The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons. Antimicrob Agents Chemother 2016; 60:2954-64. [PMID: 26926625 DOI: 10.1128/aac.00051-16] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2016] [Accepted: 02/26/2016] [Indexed: 12/30/2022] Open
Abstract
The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for chronic HCV infection.
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25
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Analysis of HCV-6 isolates among Asian-born immigrants in North America reveals their high genetic diversity and a new subtype. Virology 2016; 492:25-31. [PMID: 26896932 DOI: 10.1016/j.virol.2016.01.028] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2015] [Revised: 01/22/2016] [Accepted: 01/27/2016] [Indexed: 12/28/2022]
Abstract
We characterized full-length genomes for 15 HCV-6 isolates, all from Asian immigrants living in North America. Among these isolates, nine were novel variants showing >15% nucleotide differences from their nearest relatives, representing lineages distinct from known subtypes. The other six were classified into subtypes 6c, 6h, 6q, 6r, and 6s. The partial sequences were also determined for five additional HCV-6 isolates, three from the US and two from Canada. The latter two were assigned to new subtype 6xf as they were found to classify with two other isolates for which we recently reported their full-length genomes. We further analyzed partial Core-E1 sequences of 100 HCV-6 isolates sampled in North America, seven from the US and 93 from Canada and all from Asian immigrants except for four from Caucasians. These 100 isolates belonged to 20 assigned subtypes and 16 unclassified lineages showing great genetic diversity and enhanced significance to public health.
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Crosignani A, Riva A, Della Bella S. Analysis of peripheral blood dendritic cells as a non-invasive tool in the follow-up of patients with chronic hepatitis C. World J Gastroenterol 2016; 22:1393-1404. [PMID: 26819508 PMCID: PMC4721974 DOI: 10.3748/wjg.v22.i4.1393] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/11/2015] [Revised: 09/11/2015] [Accepted: 11/13/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) has a high propensity to establish chronic infections. Failure of HCV-infected individuals to activate effective antiviral immune responses is at least in part related to HCV-induced impairment of dendritic cells (DCs) that play a central role in activating T cell responses. Although the impact of HCV on DC phenotype and function is likely to be more prominent in the liver, major HCV-induced alterations are detectable in peripheral blood DCs (pbDCs) that represent the most accessible source of DCs. These alterations include numerical reduction, impaired production of inflammatory cytokines and increased production of immunosuppressive IL10. These changes in DCs are relevant to our understanding the immune mechanisms underlying the propensity of HCV to establish persistent infection. Importantly, the non-invasive accessibility of pbDCs renders the analysis of these cells a convenient procedure that can be serially repeated in patient follow-up. Accordingly, the study of pbDCs in HCV-infected patients during conventional treatment with pegylated interferon and ribavirin indicated that restoration of normal plasmacytoid DC count may represent an additional mechanism contributing to the efficacy of the dual therapy. It also identified the pre-treatment levels of plasmacytoid DCs and IL10 as putative predictors of response to therapy. Treatment of chronic HCV infection is changing, as new generation direct-acting antiviral agents will soon be available for use in interferon-free therapeutic strategies. The phenotypic and functional analysis of pbDCs in this novel therapeutic setting will provide a valuable tool for investigating mechanisms underlying treatment efficacy and for identifying predictors of treatment response.
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Seifert LL, Perumpail RB, Ahmed A. Update on hepatitis C: Direct-acting antivirals. World J Hepatol 2015; 7:2829-33. [PMID: 26668694 PMCID: PMC4670954 DOI: 10.4254/wjh.v7.i28.2829] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/20/2015] [Revised: 10/24/2015] [Accepted: 11/23/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) was discovered 26 years ago. For decades, interferon-based therapy has been the mainstay of treatment for HCV. Recently, several direct-acting antivirals (DAAs) have been approved for treatment of HCV-infected patients and to help combat the virus. These drugs have revolutionized the management of HCV as all-oral regimens with favorable side effect profiles and superior rates of sustained virological response. Emerging real-world data are demonstrating results comparable to registration trials for DAA agents. Suddenly, the potential for eradicating HCV is on the horizon.
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Affiliation(s)
- Leon L Seifert
- Leon L Seifert, Department of Transplantation Medicine, University Hospital Münster, 48149 Münster, Germany
| | - Ryan B Perumpail
- Leon L Seifert, Department of Transplantation Medicine, University Hospital Münster, 48149 Münster, Germany
| | - Aijaz Ahmed
- Leon L Seifert, Department of Transplantation Medicine, University Hospital Münster, 48149 Münster, Germany
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Castro R, Perazzo H, Grinsztejn B, Veloso VG, Hyde C. Chronic Hepatitis C: An Overview of Evidence on Epidemiology and Management from a Brazilian Perspective. Int J Hepatol 2015; 2015:852968. [PMID: 26693356 PMCID: PMC4677022 DOI: 10.1155/2015/852968] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/31/2015] [Revised: 11/03/2015] [Accepted: 11/11/2015] [Indexed: 02/06/2023] Open
Abstract
Chronic hepatitis C remains one of the main causes of chronic liver disease worldwide and presents a variable natural history ranging from minimal changes to advanced fibrosis and cirrhosis and its complications, such as development of hepatocellular carcinoma. Approximately, 1.45 million people are estimated to be infected by HCV in Brazil representing a major public health issue. The aim of this paper was to review the epidemiology and management of chronic hepatitis C from a Brazilian perspective. The management of chronic hepatitis C has been challenged by the use of noninvasive methods to stage liver fibrosis as an alternative to liver biopsy and the high cost of new interferon-free antiviral treatments. Moreover, the need of cost-effectiveness analysis in hepatitis C and the recent changes in treatment protocols were discussed.
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Affiliation(s)
- Rodolfo Castro
- Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation, Laboratory of Clinical Research on STD/AIDS, Avenida Brasil 4365, 21040-900 Manguinhos, RJ, Brazil
| | - Hugo Perazzo
- Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation, Laboratory of Clinical Research on STD/AIDS, Avenida Brasil 4365, 21040-900 Manguinhos, RJ, Brazil
| | - Beatriz Grinsztejn
- Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation, Laboratory of Clinical Research on STD/AIDS, Avenida Brasil 4365, 21040-900 Manguinhos, RJ, Brazil
| | - Valdilea G. Veloso
- Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation, Laboratory of Clinical Research on STD/AIDS, Avenida Brasil 4365, 21040-900 Manguinhos, RJ, Brazil
| | - Chris Hyde
- Institute of Health Research, Peninsula Technology Assessment Group (PenTAG), Evidence Synthesis and Modelling for Health Improvement (ESMI), University of Exeter Medical School, University of Exeter, St Luke's Campus, South Cloisters, Exeter EX1 2LU, UK
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Tai CJ, Li CL, Tai CJ, Wang CK, Lin LT. Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds. J Vis Exp 2015:e53124. [PMID: 26555014 DOI: 10.3791/53124] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.
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Affiliation(s)
- Chen-Jei Tai
- Department of Chinese Medicine, Taipei Medical University Hospital; Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Taipei Medical University
| | - Chia-Lin Li
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University
| | - Cheng-Jeng Tai
- Division of Hematology and Oncology, Department of Internal Medicine, Taipei Medical University Hospital; Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University
| | - Chien-Kai Wang
- Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Taipei Medical University; Division of Hematology and Oncology, Department of Internal Medicine, Taipei Medical University Hospital
| | - Liang-Tzung Lin
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University; Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University;
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High-resolution genetic profile of viral genomes: why it matters. Curr Opin Virol 2015; 14:62-70. [DOI: 10.1016/j.coviro.2015.08.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Revised: 08/07/2015] [Accepted: 08/07/2015] [Indexed: 12/12/2022]
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31
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Lindström I, Kjellin M, Palanisamy N, Bondeson K, Wesslén L, Lannergard A, Lennerstrand J. Prevalence of polymorphisms with significant resistance to NS5A inhibitors in treatment-naive patients with hepatitis C virus genotypes 1a and 3a in Sweden. Infect Dis (Lond) 2015; 47:555-62. [PMID: 25851241 DOI: 10.3109/23744235.2015.1028097] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND The future treatment of hepatitis C virus (HCV) infection will be combinations of direct-acting antivirals (DAAs) that not only target multiple viral targets, but are also effective against different HCV genotypes. Of the many drug targets in HCV, one promising target is the non-structural 5A protein (NS5A), against which inhibitors, namely daclatasvir, ledipasvir and ombitasvir, have shown potent efficacy. However, since HCV is known to have very high sequence diversity, development of resistance is a problem against but not limited to NS5A inhibitors (i.e. resistance also found against NS3-protease and NS5B non-nucleoside inhibitors), when used in suboptimal combinations. Furthermore, it has been shown that natural resistance against DAAs is present in treatment-naïve patients and such baseline resistance will potentially complicate future treatment strategies. METHODS A pan-genotypic population-sequencing method with degenerated primers targeting the NS5A region was developed. We have investigated the prevalence of baseline resistant variants in 127 treatment-naïve patients of HCV genotypes 1a, 1b, 2b and 3a. RESULTS The method could successfully sequence more than 95% of genotype 1a, 1b and 3a samples. Interpretation of fold resistance data against the NS5A inhibitors was done with the help of earlier published phenotypic data. Baseline resistance variants associated with high resistance (1000-50,000-fold) was found in three patients: Q30H or Y93N in genotype 1a patients and further Y93H in a genotype 3a patient. CONCLUSION Using this method, baseline resistance can be examined and the data could have a potential role in selecting the optimal and cost-efficient treatment for the patient.
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Affiliation(s)
- Ida Lindström
- From the 1 Clinical Virology, Department of Medical Sciences, Uppsala University , Uppsala
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AP-2-associated protein kinase 1 and cyclin G-associated kinase regulate hepatitis C virus entry and are potential drug targets. J Virol 2015; 89:4387-404. [PMID: 25653444 DOI: 10.1128/jvi.02705-14] [Citation(s) in RCA: 103] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
UNLABELLED Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the μ subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved anticancer drugs sunitinib and erlotinib, could block HCV assembly. Here, we hypothesized that AAK1 and GAK regulate HCV entry independently of their effect on HCV assembly. Indeed, silencing AAK1 and GAK expression inhibited entry of pseudoparticles and cell culture grown-HCV and internalization of Dil-labeled HCV particles with no effect on HCV attachment or RNA replication. AAK1 or GAK depletion impaired epidermal growth factor (EGF)-mediated enhanced HCV entry and endocytosis of EGF receptor (EGFR), an HCV entry cofactor and erlotinib's cancer target. Moreover, either RNA interference-mediated depletion of AP2M1 or NUMB, each a substrate of AAK1 and/or GAK, or overexpression of either an AP2M1 or NUMB phosphorylation site mutant inhibited HCV entry. Last, in addition to affecting assembly, sunitinib and erlotinib inhibited HCV entry at a postbinding step, their combination was synergistic, and their antiviral effect was reversed by either AAK1 or GAK overexpression. Together, these results validate AAK1 and GAK as critical regulators of HCV entry that function in part by activating EGFR, AP2M1, and NUMB and as the molecular targets underlying the antiviral effect of sunitinib and erlotinib (in addition to EGFR), respectively. IMPORTANCE Understanding the host pathways hijacked by HCV is critical for developing host-centered anti-HCV approaches. Entry represents a potential target for antiviral strategies; however, no FDA-approved HCV entry inhibitors are currently available. We reported that two host kinases, AAK1 and GAK, regulate HCV assembly. Here, we provide evidence that AAK1 and GAK regulate HCV entry independently of their role in HCV assembly and define the mechanisms underlying AAK1- and GAK-mediated HCV entry. By regulating temporally distinct steps in the HCV life cycle, AAK1 and GAK represent "master regulators" of HCV infection and potential targets for antiviral strategies. Indeed, approved anticancer drugs that potently inhibit AAK1 or GAK inhibit HCV entry in addition to assembly. These results contribute to an understanding of the mechanisms of HCV entry and reveal attractive host targets for antiviral strategies as well as approved candidate inhibitors of these targets, with potential implications for other viruses that hijack clathrin-mediated pathways.
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Maiti M, Maiti M, Rozenski J, De Jonghe S, Herdewijn P. Aspartic acid based nucleoside phosphoramidate prodrugs as potent inhibitors of hepatitis C virus replication. Org Biomol Chem 2015; 13:5158-74. [DOI: 10.1039/c5ob00427f] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
A series of novel nucleoside phosphoramidate protides has been synthesized and shown as potent inhibitors of hepatitis C virus replication. The conjugates are having a diverse structural variation in the promoiety part and can be catalytically processed to deliver active nucleotides.
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Affiliation(s)
- Munmun Maiti
- Rega Institute for Medical Research
- Medicinal Chemistry
- KU Leuven
- 3000 Leuven
- Belgium
| | - Mohitosh Maiti
- Rega Institute for Medical Research
- Medicinal Chemistry
- KU Leuven
- 3000 Leuven
- Belgium
| | - Jef Rozenski
- Rega Institute for Medical Research
- Medicinal Chemistry
- KU Leuven
- 3000 Leuven
- Belgium
| | - Steven De Jonghe
- Rega Institute for Medical Research
- Medicinal Chemistry
- KU Leuven
- 3000 Leuven
- Belgium
| | - Piet Herdewijn
- Rega Institute for Medical Research
- Medicinal Chemistry
- KU Leuven
- 3000 Leuven
- Belgium
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Sølund C, Krarup H, Ramirez S, Thielsen P, Røge BT, Lunding S, Barfod TS, Madsen LG, Tarp B, Christensen PB, Gerstoft J, Laursen AL, Bukh J, Weis N. Nationwide experience of treatment with protease inhibitors in chronic hepatitis C patients in Denmark: identification of viral resistance mutations. PLoS One 2014; 9:e113034. [PMID: 25438153 PMCID: PMC4249835 DOI: 10.1371/journal.pone.0113034] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2014] [Accepted: 10/18/2014] [Indexed: 12/15/2022] Open
Abstract
Background and Aims The first standard of care in treatment of chronic HCV genotype 1 infection involving directly acting antivirals was protease inhibitors telaprevir or boceprevir combined with pegylated-interferon and ribavirin (triple therapy). Phase III studies include highly selected patients. Thus, treatment response and development of viral resistance during triple therapy in a routine clinical setting needs to be determined. The aims of this study were to investigate treatment outcome and identify sequence variations after triple therapy in patients with chronic HCV genotype 1 infection in a routine clinical setting. Methods 80 patients, who initiated and completed triple therapy in Denmark between May 2011 and November 2012, were included. Demographic data and treatment response were obtained from the Danish Database for Hepatitis B and C. Direct sequencing and clonal analysis of the RT-PCR amplified NS3 protease were performed in patients without cure following triple therapy. Results 38 (47%) of the patients achieved cure, 15 (19%) discontinued treatment due to adverse events and remained infected, and 27 (34%) experienced relapse or treatment failure of whom 15 of 21 analyzed patients had well-described protease inhibitor resistance variants detected. Most frequently detected protease variants were V36M and/or R155K, and V36M, in patients with genotype 1a and 1b infection, respectively. Conclusions The cure rate after triple therapy in a routine clinical setting was 47%, which is substantially lower than in clinical trials. Resistance variants towards protease inhibitors were seen in 71% of patients failing therapy indicating that resistance could have an important role in treatment response.
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Affiliation(s)
- Christina Sølund
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark; and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Henrik Krarup
- Section of Molecular Diagnostics, Clinical Biochemistry and Department of Medical Gastroenterology, Aalborg University Hospital, Aalborg, Denmark
| | - Santseharay Ramirez
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark; and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Peter Thielsen
- Department of Gastroenterology, Copenhagen University Hospital, Herlev, Denmark
| | - Birgit T. Røge
- Department of Medicine, Kolding Hospital, Kolding, Denmark
| | - Suzanne Lunding
- Department of Infectious Diseases, Copenhagen University Hospital, Hillerød, Denmark
| | - Toke S. Barfod
- Department of Infectious Diseases, Roskilde Hospital, Roskilde, Denmark
| | - Lone G. Madsen
- Department of Gastroenterology, Køge Hospital, Køge, Denmark
| | - Britta Tarp
- Diagnostic Center, Silkeborg Regional Hospital, Silkeborg, Denmark
| | - Peer B. Christensen
- Department of Infectious Diseases, Odense University Hospital, Odense, Denmark
| | - Jan Gerstoft
- Department of Infectious Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark
| | - Alex L. Laursen
- Department of Infectious Diseases, Aarhus University Hospital, Skejby, Aarhus, Denmark
| | - Jens Bukh
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark; and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Nina Weis
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- * E-mail:
| | - The DANHEP group
- Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
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Peres-da-Silva A, de Almeida AJ, Lampe E. NS5A inhibitor resistance-associated polymorphisms in Brazilian treatment-naive patients infected with genotype 1 hepatitis C virus. J Antimicrob Chemother 2014; 70:726-30. [PMID: 25414201 DOI: 10.1093/jac/dku462] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
OBJECTIVES Several promising NS5A protein inhibitors for hepatitis C virus (HCV) treatment, showing good antiviral activity, are currently being evaluated in clinical trials. However, viral breakthroughs associated with resistant variants have been observed, especially in patients infected with HCV-1a. We aimed to evaluate the occurrence of potential resistance mutations in the NS5A gene of HCV among Brazilian treatment-naive patients. METHODS Direct sequencing of the HCV NS5A gene was performed in serum samples of 106 treatment-naive patients infected with subtypes 1a (n = 52) and 1b (n = 54). The sequence variability, signature patterns in amino acid sequences and variants associated with NS5A inhibitors were evaluated. RESULTS The M28T and Y93H mutations were found in the subtype 1a sequences of two (3.85%) patients, and seven (13.46%) other patients presented the secondary mutation(s) H58P, E62D or H58P-E62D. For subtype 1b, the Y93H mutation was found in two (3.70%) patients and the substitutions R30Q, L31M, P58S and I280V were found in eight (14.81%) patients. Two distinct HCV-1a clades were distinguished by a phylogenetic analysis performed along with representative HCV-1a sequences and sequences containing HCV NS5A inhibitor resistance mutations retrieved from the Los Alamos database. All Brazilian sequences formed a large group of related sequences inside clade 1. It is noteworthy that 65.85% of sequences with substitution at sites 28, 30, 31 and 93 were found in clade 1. CONCLUSION Brazilian HCV-1a sequences presented a peculiar pattern of amino acid composition, mutations and frequencies, which is distinct from other previously characterized sequences from other locations. The association of these findings with the outcome of treatment with NS5A inhibitors awaits further analysis.
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Affiliation(s)
- Allan Peres-da-Silva
- Laboratório de Hepatites Virais, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, RJ, Brazil
| | - Adilson José de Almeida
- Laboratório de Hepatites Virais, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, RJ, Brazil Hospital Universitário Gaffrée e Guinle/UNIRIO, Rio de Janeiro, RJ, Brazil
| | - Elisabeth Lampe
- Laboratório de Hepatites Virais, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, RJ, Brazil
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Munteanu DI, Rockstroh JK. New agents for the treatment of hepatitis C in patients co-infected with HIV. Ther Adv Infect Dis 2014; 1:71-80. [PMID: 25165545 DOI: 10.1177/2049936113479591] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Pilot trials evaluating the efficacy and safety of the first licensed hepatitis C virus (HCV) protease inhibitors (PIs), boceprevir (BOC) and telaprevir (TVR), for the treatment of genotype 1 infection in HCV/HIV co-infected patients revealed similar results as in HCV mono-infected patients. HCV liver disease progresses more rapidly in co-infected patients, particularly with advanced immunodeficiency. Therefore, HCV treatment in HIV is of great importance. However, dual therapy with pegylated interferon (PegIFN) and ribavirin (RBV) has been associated with lower cure rates and increased toxicities in co-infected subjects, thereby limiting overall HCV therapy uptake. The availability of HCV PIs opens new perspectives for HCV cure in co-infected patients, with a 70% sustained virologic response (SVR) rate in HCV treatment-naïve patients. Despite these impressive advances, the use of the new treatment options has been low, reflecting the complex issues with modern triple HCV therapy. Indeed pill burden, adverse events (AEs), drug-drug interactions (DDIs) and high costs complicate HCV therapy in HIV. So far, studies have shown no tolerability differences in mono- and co-infected patients with the early stages of liver fibrosis. Regarding DDIs between HVC PIs and antiretroviral drugs, TVR can be safely administered with efavirenz (with dose adjustment of TVR), etravirine (ETR), rilpivirine, boosted atazanavir (ATV/r) and raltegravir (RAL), while BOC can be safely administered with ETR, RAL and potentially ATV/r for treatment-naïve patients under careful monitoring. Currently, the great number of HCV molecules under development is promising substantially improved treatment paradigms with shorter treatment durations, fewer AEs, less DDIs, once-daily administration and even interferon-free regimens. The decision to treat now with the available HCV PIs or defer therapy until the second generation of HCV direct acting antivirals become available should be based on liver fibrosis staging and fibrosis progression during follow up. More data are urgently needed regarding the efficacy of triple therapy in HIV/HCV co-infected patients who previously failed PegIFN/RBV therapy as well as in patients with more advanced fibrosis stages.
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Affiliation(s)
- Daniela I Munteanu
- Department of Medicine I, Bonn University Hospital, Bonn, Germany and Adults III Department, Matei Bals National Institute of Infectious Diseases, Bucharest, Romania
| | - Jürgen K Rockstroh
- Department of Medicine I, Bonn University Hospital, Sigmund-Freud-Str. 25, Bonn 53105, Germany
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Lee JC, Tseng CK, Young KC, Sun HY, Wang SW, Chen WC, Lin CK, Wu YH. Andrographolide exerts anti-hepatitis C virus activity by up-regulating haeme oxygenase-1 via the p38 MAPK/Nrf2 pathway in human hepatoma cells. Br J Pharmacol 2014; 171:237-52. [PMID: 24117426 DOI: 10.1111/bph.12440] [Citation(s) in RCA: 126] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2013] [Revised: 09/16/2013] [Accepted: 09/24/2013] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND AND PURPOSE This study aimed to evaluate the anti-hepatitis C virus (HCV) activity of andrographolide, a diterpenoid lactone extracted from Andrographis paniculata, and to identify the signalling pathway involved in its antiviral action. EXPERIMENTAL APPROACH Using HCV replicon and HCVcc infectious systems, we identified anti-HCV activity of andrographolide by measuring protein and RNA levels. A reporter activity assay was used to determine transcriptional regulation of anti-HCV agents. A specific inhibitor and short hairpin RNAs were used to investigate the mechanism responsible for the effect of andrographolide on HCV replication. KEY RESULTS In HCV replicon and HCVcc infectious systems, andrographolide time- and dose-dependently suppressed HCV replication. When combined with IFN-α, an inhibitor targeting HCV NS3/4A protease (telaprevir), or NS5B polymerase (PSI-7977), andrographolide exhibited a significant synergistic effect. Andrographolide up-regulated the expression of haeme oxygenase-1 (HO-1), leading to increased amounts of its metabolite biliverdin, which was found to suppress HCV replication by promoting the antiviral IFN responses and inhibiting NS3/4A protease activity. Significantly, these antiviral effects were attenuated by an HO-1-specific inhibitor or HO-1 gene knockdown, indicating that HO-1 contributed to the anti-HCV activity of andrographolide. Andrographolide activated p38 MAPK phosphorylation, which stimulated nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated HO-1 expression, and this was found to be associated with its anti-HCV activity. CONCLUSIONS AND IMPLICATIONS Our results demonstrate that andrographolide has the potential to control HCV replication and suggest that targeting the Nrf2-HO-1 signalling pathway might be a promising strategy for drug development.
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Affiliation(s)
- Jin-Ching Lee
- Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan; Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
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Performance comparison of the versant HCV genotype 2.0 assay (LiPA) and the abbott realtime HCV genotype II assay for detecting hepatitis C virus genotype 6. J Clin Microbiol 2014; 52:3685-92. [PMID: 25100817 DOI: 10.1128/jcm.00882-14] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5' untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1.
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Poveda E, Wyles DL, Mena A, Pedreira JD, Castro-Iglesias A, Cachay E. Update on hepatitis C virus resistance to direct-acting antiviral agents. Antiviral Res 2014; 108:181-91. [PMID: 24911972 DOI: 10.1016/j.antiviral.2014.05.015] [Citation(s) in RCA: 152] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2014] [Revised: 05/13/2014] [Accepted: 05/22/2014] [Indexed: 12/12/2022]
Abstract
Resistance to direct-acting antiviral (DAA) agents against hepatitis C virus (HCV) infection is driven by the selection of mutations at different positions in the NS3 protease, NS5B polymerase and NS5A proteins. With the exception of NS5B nucleos(t)ide inhibitors, most DAAs possess a low genetic barrier to resistance, with significant cross-resistance between compounds belonging to the same family. However, a specific mutation profile is associated with each agent or drug class and varies depending on the genotype/subtype (e.g., genotype 1b showed higher rates of sustained virological response (SVR) and a higher genetic barrier for resistance than genotype 1a). Moreover, some resistance mutations exist as natural polymorphisms in certain genotypes/subtypes at frequencies that require baseline drug resistance testing before recommending certain antivirals. For example, the polymorphism Q80K is frequently found among genotype 1a (19-48%) and is associated with resistance to simeprevir. Similarly, L31M and Y93H, key resistance mutations to NS5A inhibitors, are frequently found (6-12%) among NS5A genotype 1 sequences. In particular, the presence of these polymorphisms may be of relevance in poorly interferon-responsive patients (i.e., null responders and non-CC IL28B) under DAA-based therapies in combination with pegylated interferon-α plus ribavirin. The relevance of pre-existing resistance mutations for responses to interferon-free DAA therapies is unclear for most regimens and requires further study.
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Affiliation(s)
- Eva Poveda
- Grupo de Virología Clínica, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Spain.
| | - David L Wyles
- Department of Medicine, Owen Clinic and Division of Infectious Diseases, UC San Diego, USA
| | - Alvaro Mena
- Grupo de Virología Clínica, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Spain
| | - José D Pedreira
- Grupo de Virología Clínica, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Spain
| | - Angeles Castro-Iglesias
- Grupo de Virología Clínica, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Spain
| | - Edward Cachay
- Department of Medicine, Owen Clinic and Division of Infectious Diseases, UC San Diego, USA
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Prevalence of hepatitis C virus subgenotypes 1a and 1b in Japanese patients: ultra-deep sequencing analysis of HCV NS5B genotype-specific region. PLoS One 2013; 8:e73615. [PMID: 24069214 PMCID: PMC3775835 DOI: 10.1371/journal.pone.0073615] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2013] [Accepted: 07/20/2013] [Indexed: 01/13/2023] Open
Abstract
Background Hepatitis C virus (HCV) subgenotypes 1a and 1b have different impacts on the treatment response to peginterferon plus ribavirin with direct-acting antivirals (DAAs) against patients infected with HCV genotype 1, as the emergence rates of resistance mutations are different between these two subgenotypes. In Japan, almost all of HCV genotype 1 belongs to subgenotype 1b. Methods and Findings To determine HCV subgenotype 1a or 1b in Japanese patients infected with HCV genotype 1, real-time PCR-based method and Sanger method were used for the HCV NS5B region. HCV subgenotypes were determined in 90% by real-time PCR-based method. We also analyzed the specific probe regions for HCV subgenotypes 1a and 1b using ultra-deep sequencing, and uncovered mutations that could not be revealed using direct-sequencing by Sanger method. We estimated the prevalence of HCV subgenotype 1a as 1.2-2.5% of HCV genotype 1 patients in Japan. Conclusions Although real-time PCR-based HCV subgenotyping method seems fair for differentiating HCV subgenotypes 1a and 1b, it may not be sufficient for clinical practice. Ultra-deep sequencing is useful for revealing the resistant strain(s) of HCV before DAA treatment as well as mixed infection with different genotypes or subgenotypes of HCV.
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Lin LT, Chen TY, Lin SC, Chung CY, Lin TC, Wang GH, Anderson R, Lin CC, Richardson CD. Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry. BMC Microbiol 2013; 13:187. [PMID: 23924316 PMCID: PMC3750913 DOI: 10.1186/1471-2180-13-187] [Citation(s) in RCA: 134] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2012] [Accepted: 07/31/2013] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND We previously identified two hydrolyzable tannins, chebulagic acid (CHLA) and punicalagin (PUG) that blocked herpes simplex virus type 1 (HSV-1) entry and spread. These compounds inhibited viral glycoprotein interactions with cell surface glycosaminoglycans (GAGs). Based on this property, we evaluated their antiviral efficacy against several different viruses known to employ GAGs for host cell entry. RESULTS Extensive analysis of the tannins' mechanism of action was performed on a panel of viruses during the attachment and entry steps of infection. Virus-specific binding assays and the analysis of viral spread during treatment with these compounds were also conducted. CHLA and PUG were effective in abrogating infection by human cytomegalovirus (HCMV), hepatitis C virus (HCV), dengue virus (DENV), measles virus (MV), and respiratory syncytial virus (RSV), at μM concentrations and in dose-dependent manners without significant cytotoxicity. Moreover, the natural compounds inhibited viral attachment, penetration, and spread, to different degrees for each virus. Specifically, the tannins blocked all these steps of infection for HCMV, HCV, and MV, but had little effect on the post-fusion spread of DENV and RSV, which could suggest intriguing differences in the roles of GAG-interactions for these viruses. CONCLUSIONS CHLA and PUG may be of value as broad-spectrum antivirals for limiting emerging/recurring viruses known to engage host cell GAGs for entry. Further studies testing the efficacy of these tannins in vivo against certain viruses are justified.
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Affiliation(s)
- Liang-Tzung Lin
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
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Implications of baseline polymorphisms for potential resistance to NS3 protease inhibitors in Hepatitis C virus genotypes 1a, 2b and 3a. Antiviral Res 2013; 99:12-7. [DOI: 10.1016/j.antiviral.2013.04.018] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2013] [Revised: 04/12/2013] [Accepted: 04/23/2013] [Indexed: 02/07/2023]
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Newman RM, Kuntzen T, Weiner B, Berical A, Charlebois P, Kuiken C, Murphy DG, Simmonds P, Bennett P, Lennon NJ, Birren BW, Zody MC, Allen TM, Henn MR. Whole genome pyrosequencing of rare hepatitis C virus genotypes enhances subtype classification and identification of naturally occurring drug resistance variants. J Infect Dis 2013; 208:17-31. [PMID: 23136221 PMCID: PMC3666132 DOI: 10.1093/infdis/jis679] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2012] [Accepted: 08/08/2012] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Infection with hepatitis C virus (HCV) is a burgeoning worldwide public health problem, with 170 million infected individuals and an estimated 20 million deaths in the coming decades. While 6 main genotypes generally distinguish the global geographic diversity of HCV, a multitude of closely related subtypes within these genotypes are poorly defined and may influence clinical outcome and treatment options. Unfortunately, the paucity of genetic data from many of these subtypes makes time-consuming primer walking the limiting step for sequencing understudied subtypes. METHODS Here we combined long-range polymerase chain reaction amplification with pyrosequencing for a rapid approach to generate the complete viral coding region of 31 samples representing poorly defined HCV subtypes. RESULTS Phylogenetic classification based on full genome sequences validated previously identified HCV subtypes, identified a recombinant sequence, and identified a new distinct subtype of genotype 4. Unlike conventional sequencing methods, use of deep sequencing also facilitated characterization of minor drug resistance variants within these uncommon or, in some cases, previously uncharacterized HCV subtypes. CONCLUSIONS These data aid in the classification of uncommon HCV subtypes while also providing a high-resolution view of viral diversity within infected patients, which may be relevant to the development of therapeutic regimens to minimize drug resistance.
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Affiliation(s)
- Ruchi M Newman
- Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA.
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Jiang Y, Andrews SW, Condroski KR, Buckman B, Serebryany V, Wenglowsky S, Kennedy AL, Madduru MR, Wang B, Lyon M, Doherty GA, Woodard BT, Lemieux C, Geck Do M, Zhang H, Ballard J, Vigers G, Brandhuber BJ, Stengel P, Josey JA, Beigelman L, Blatt L, Seiwert SD. Discovery of danoprevir (ITMN-191/R7227), a highly selective and potent inhibitor of hepatitis C virus (HCV) NS3/4A protease. J Med Chem 2013; 57:1753-69. [PMID: 23672640 DOI: 10.1021/jm400164c] [Citation(s) in RCA: 109] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
HCV serine protease NS3 represents an attractive drug target because it is not only essential for viral replication but also implicated in the viral evasion of the host immune response pathway through direct cleavage of key proteins in the human innate immune system. Through structure-based drug design and optimization, macrocyclic peptidomimetic molecules bearing both a lipophilic P2 isoindoline carbamate and a P1/P1' acylsulfonamide/acylsulfamide carboxylic acid bioisostere were prepared that possessed subnanomolar potency against the NS3 protease in a subgenomic replicon-based cellular assay (Huh-7). Danoprevir (compound 49) was selected as the clinical development candidate for its favorable potency profile across multiple HCV genotypes and key mutant strains and for its good in vitro ADME profiles and in vivo target tissue (liver) exposures across multiple animal species. X-ray crystallographic studies elucidated several key features in the binding of danoprevir to HCV NS3 protease and proved invaluable to our iterative structure-based design strategy.
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Affiliation(s)
- Yutong Jiang
- Array BioPharma , 3200 Walnut Street, Boulder, Colorado 80301, United States
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Gising J, Belfrage AK, Alogheli H, Ehrenberg A, Åkerblom E, Svensson R, Artursson P, Karlén A, Danielson UH, Larhed M, Sandström A. Achiral pyrazinone-based inhibitors of the hepatitis C virus NS3 protease and drug-resistant variants with elongated substituents directed toward the S2 pocket. J Med Chem 2013; 57:1790-801. [PMID: 23517538 DOI: 10.1021/jm301887f] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Herein we describe the design, synthesis, inhibitory potency, and pharmacokinetic properties of a novel class of achiral peptidomimetic HCV NS3 protease inhibitors. The compounds are based on a dipeptidomimetic pyrazinone glycine P3P2 building block in combination with an aromatic acyl sulfonamide in the P1P1' position. Structure-activity relationship data and molecular modeling support occupancy of the S2 pocket from elongated R(6) substituents on the 2(1H)-pyrazinone core and several inhibitors with improved inhibitory potency down to Ki = 0.11 μM were identified. A major goal with the design was to produce inhibitors structurally dissimilar to the di- and tripeptide-based HCV protease inhibitors in advanced stages of development for which cross-resistance might be an issue. Therefore, the retained and improved inhibitory potency against the drug-resistant variants A156T, D168V, and R155K further strengthen the potential of this class of inhibitors. A number of the inhibitors were tested in in vitro preclinical profiling assays to evaluate their apparent pharmacokinetic properties. The various R(6) substituents were found to have a major influence on solubility, metabolic stability, and cell permeability.
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Affiliation(s)
- Johan Gising
- Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, BMC, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden
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Kim DY, Kim IH, Jeong SH, Cho YK, Lee JH, Jin YJ, Lee D, Suh DJ, Han KH, Park NH, Kang HY, Jung YK, Kim YS, Kim KA, Lee YJ, Lee BS, Yim HJ, Lee HJ, Baik SK, Tak WY, Lee SJ, Chung WJ, Choi SK, Cho EY, Heo J, Kim DJ, Song BC, Kim MW, Lee J, Chae HB, Choi DH, Choi HY, Ki M. A nationwide seroepidemiology of hepatitis C virus infection in South Korea. Liver Int 2013; 33:586-594. [PMID: 23356674 DOI: 10.1111/liv.12108] [Citation(s) in RCA: 75] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2012] [Accepted: 12/19/2012] [Indexed: 12/13/2022]
Abstract
BACKGROUND & AIMS The aim of this study was to reveal nationwide seroprevalence of HCV infection in South Korea by a large-scale survey. METHODS From January to December 2009, a total of 291 314 adults underwent health check-up in 29 centres nationwide. The data concerning anti-HCV antibody and biochemical tests were obtained from all participants. Among subjects with positive anti-HCV, such data as HCV RNA, genotypes and treatment detail were additionally analysed. RESULTS Using an estimated 2009 population of Korea, the age, sex and area-adjusted anti-HCV positive rate was 0.78%. Anti-HCV prevalence in female patients (0.83%) was higher than that in male patients (0.75%). Gradual increase in anti-HCV positivity was observed, from 0.34% in those aged 20-29 years to 2.31% in those >70 years. The age- and sex-adjusted anti-HCV prevalence varied in different areas, being higher in Busan and Jeonnam (1.53-2.07%), mid-level in Seoul and surrounding districts (0.50-0.61%) and lower in Jeju (0.23%). The comparative analysis of laboratory variables between anti-HCV (+) and anti-HCV (-) group revealed significantly higher levels of alanine aminotransferase and lower levels of serum lipids in anti-HCV (+) group. Among 1 718 anti-HCV positive subjects, serum HCV RNA was measured only in 478 people, of whom 268 (56.1%) patients had detectable HCV RNA in serum. Among 50 patients for whom assessment of response to antiviral therapy was feasible, overall sustained virological response was achieved in 84% of patients. CONCLUSION The prevalence of HCV infection is low in South Korea. Studies to analyse risk factors are warranted to reduce HCV infection.
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Affiliation(s)
- Do Young Kim
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea
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Salvatierra K, Fareleski S, Forcada A, López-Labrador FX. Hepatitis C virus resistance to new specifically-targeted antiviral therapy: A public health perspective. World J Virol 2013; 2:6-15. [PMID: 24175225 PMCID: PMC3785043 DOI: 10.5501/wjv.v2.i1.6] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/07/2012] [Revised: 12/30/2012] [Accepted: 01/17/2013] [Indexed: 02/05/2023] Open
Abstract
Until very recently, treatment for chronic hepatitis C virus (HCV) infection has been based on the combination of two non-viral specific drugs: pegylated interferon-α and ribavirin, which is effective in, overall, about 40%-50% of cases. To improve the response to treatment, novel drugs have been designed to specifically block viral proteins. Multiple compounds are under development, and the approval for clinical use of the first of such direct-acting antivirals in 2011 (Telaprevir and Boceprevir), represents a milestone in HCV treatment. HCV therapeutics is entering a new expanding era, and a highly-effective cure is envisioned for the first time since the discovery of the virus in 1989. However, any antiviral treatment may be limited by the capacity of the virus to overcome the selective pressure of new drugs, generating antiviral resistance. Here, we try to provide a basic overview of new treatments, HCV resistance to new antivirals and some considerations derived from a Public Health perspective, using HCV resistance to protease and polymerase inhibitors as examples.
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Affiliation(s)
- Karina Salvatierra
- Karina Salvatierra, Sabrina Fareleski, F Xavier López-Labrador, Joint Unit in Genomics and Health, Centre for Public Health Research, Public Health Department, Generalitat Valenciana/Institut Cavanilles, University of Valencia, 46020 Valencia, Spain
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Fukazawa H, Suzuki T, Wakita T, Murakami Y. A cell-based, microplate colorimetric screen identifies 7,8-benzoflavone and green tea gallate catechins as inhibitors of the hepatitis C virus. Biol Pharm Bull 2012; 35:1320-7. [PMID: 22863932 DOI: 10.1248/bpb.b12-00251] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We describe a cell-based, microplate colorimetric screen for anti-hepatitis C virus (HCV) drugs that exploits the HCV-JFH1 viral culture system. Antiviral activity was assessed by measuring protection against the HCV-JFH1-induced cytopathic effect (CPE) in Huh7.5.1 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay. The use of serum-free medium substantially sensitized Huh7.5.1 cells to HCV-induced CPE, causing sufficient cell death to perform colorimetric assays for anti-HCV activity in 96-well plates. As a proof of concept, we carried out a pilot screen of an inhibitor library and identified cyclosporin A and tamoxifen, two compounds with reported anti-HCV activity. Using the assay, we discovered the anti-HCV properties of the plant flavonoids epigallocatechin gallate (EGCG) and 7,8-benzoflavone (α-naphthoflavone). Other gallate-type catechins and flavones also displayed anti-HCV activity, but 5,6-benzoflavone (β-naphthoflavone), flavanone, and non-gallate catechins were inactive. EGCG apparently acted mainly on HCV entry, although it may also block other steps. In contrast, 7,8-benzoflavone was presumed to inhibit later stages of the HCV life cycle. This assay is simple, reliable and cost-effective; does not require any specially engineered cell lines or viruses; and should be useful in the identification of compounds with anti-HCV activity.
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Affiliation(s)
- Hidesuke Fukazawa
- Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan.
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Treatment of Hepatitis C in Patients Infected with Human Immunodeficiency Virus in the Direct-Acting Antiviral Era. Infect Dis Clin North Am 2012; 26:931-48. [DOI: 10.1016/j.idc.2012.08.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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