RESUMEN
BACKGROUND & AIMS: Retreatment with glecaprevir/pibrentasvir (G/P) resulted in a rate of sustained virologic response 12 weeks after treatment completion (SVR12) of >90% in HCV genotype 1 (GT1) patients who previously failed a regimen of sofosbuvir plus an NS5A inhibitor (NS5Ai). This study investigated the prevalence and impact of baseline NS3 and NS5A resistance-associated substitutions (RASs) on the efficacy of G/P in prior GT1 sofosbuvir+NS5Ai failures and the persistence of treatment-emergent RASs. METHODS: Longitudinal samples from 177 patients enrolled in a phase IIIb, randomized pragmatic clinical trial were analyzed. Patients without cirrhosis were randomized to 12 or 16 weeks of G/P, and patients with compensated cirrhosis were randomized to G/P and ribavirin for 12 weeks or G/P for 16 weeks. Linkage of RAS was identified using Primer-ID next-generation sequencing at a 15% cut-off. RESULTS: Of 177 patients, 169 (95.5%) were PI-naïve. All 33 GT1b-infected patients achieved SVR12. In GT1a-infected patients, baseline NS5A RASs were prevalent (74.5%, 105/141) but NS3 RASs were uncommon. Baseline NS3 RASs had no impact on G/P efficacy and patients with baseline NS5A RASs showed a numerically but not statistically significantly lower SVR12 rate compared to those without NS5A RASs (89% vs. 97%). SVR12 was achieved in 34 of 35 (97%) patients without NS5A baseline substitution, and 53 of 57 (93%), 35 of 40 (88%), 5 of 8 (63%) with single, double-linked, and triple-linked NS5A substitutions, respectively. Among 13 patients with virologic failure, 4 acquired treatment-emergent NS3 RASs and 10 acquired NS5A RASs. CONCLUSION: Baseline NS5A RASs were highly prevalent. The presence of an increasing number of linked NS5A RASs in GT1a showed a trend in decreasing SVR12 rates, although no specific NS5A RASs or their linkage pattern were associated with lower SVR12 rates. LAY SUMMARY: Direct-acting antivirals have revolutionized the treatment of chronic hepatitis C infection, but treatment failure occurs in some patients. Retreatment of patients who previously failed a regimen consisting of sofosbuvir and an NS5A inhibitor with a regimen of glecaprevir and pibrentasvir (G/P) is >90% effective. Herein, we analyzed samples from these patients and showed that retreatment efficacy with G/P is lower in patients with double- or triple-linked NS5A resistance mutations than in patients with single or no NS5A resistance mutations. CLINICAL TRIAL NUMBER: NCT03092375.
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Bencimidazoles/farmacología , Resistencia a Medicamentos/inmunología , Pirrolidinas/farmacología , Quinoxalinas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Sofosbuvir/metabolismo , Sulfonamidas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Adulto , Antivirales/administración & dosificación , Antivirales/metabolismo , Bencimidazoles/uso terapéutico , Combinación de Medicamentos , Femenino , Hepatitis C/tratamiento farmacológico , Hepatitis C/epidemiología , Hepatitis C/fisiopatología , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Pirrolidinas/uso terapéutico , Quinoxalinas/administración & dosificación , Quinoxalinas/uso terapéutico , ARN Polimerasa Dependiente del ARN/farmacología , Sofosbuvir/administración & dosificación , Sulfonamidas/uso terapéutico , Estados Unidos/epidemiología , Proteínas no Estructurales Virales/farmacologíaAsunto(s)
Antivirales/farmacocinética , Carbamatos/farmacocinética , Imidazoles/farmacocinética , Intercambio Materno-Fetal , Placenta/metabolismo , Pirrolidinas/farmacocinética , Uridina/análogos & derivados , Valina/análogos & derivados , Antivirales/metabolismo , Carbamatos/metabolismo , Femenino , Hepatitis C/tratamiento farmacológico , Humanos , Imidazoles/metabolismo , Embarazo , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Pirrolidinas/metabolismo , Sofosbuvir/metabolismo , Sofosbuvir/farmacocinética , Uridina/metabolismo , Uridina/farmacocinética , Valina/metabolismo , Valina/farmacocinéticaRESUMEN
SARS-CoV-2 is responsible for the current COVID-19 pandemic. On the basis of our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues (the triphosphates of Sofosbuvir, Alovudine, and AZT) inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). We also demonstrated that a library of additional nucleotide analogues terminate RNA synthesis catalyzed by the SARS-CoV-2 RdRp, a well-established drug target for COVID-19. Here, we used polymerase extension experiments to demonstrate that the active triphosphate form of Sofosbuvir (an FDA-approved hepatitis C drug) is incorporated by SARS-CoV-2 RdRp and blocks further incorporation. Using the molecular insight gained from the previous studies, we selected the active triphosphate forms of six other antiviral agents, Alovudine, Tenofovir alafenamide, AZT, Abacavir, Lamivudine, and Emtricitabine, for evaluation as inhibitors of the SARS-CoV-2 RdRp and demonstrated the ability of these viral polymerase inhibitors to be incorporated by SARS-CoV-2 RdRp, where they terminate further polymerase extension with varying efficiency. These results provide a molecular basis for inhibition of the SARS-CoV-2 RdRp by these nucleotide analogues. If sufficient efficacy of some of these FDA-approved drugs in inhibiting viral replication in cell culture is established, they may be explored as potential COVID-19 therapeutics.
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Antivirales , Betacoronavirus , ARN Polimerasa Dependiente del ARN , Proteínas no Estructurales Virales , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Betacoronavirus/enzimología , Betacoronavirus/genética , COVID-19 , Infecciones por Coronavirus/virología , Didesoxinucleósidos/química , Didesoxinucleósidos/metabolismo , Didesoxinucleósidos/farmacología , Humanos , Pandemias , Neumonía Viral/virología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2 , Sofosbuvir/química , Sofosbuvir/metabolismo , Sofosbuvir/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoAsunto(s)
Antivirales/uso terapéutico , Carbamatos/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Neumonía Viral/tratamiento farmacológico , Sofosbuvir/uso terapéutico , Antivirales/química , Antivirales/metabolismo , Betacoronavirus/aislamiento & purificación , COVID-19 , Infecciones por Coronavirus/virología , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Quimioterapia Combinada , Humanos , Pandemias , Neumonía Viral/virología , SARS-CoV-2 , Sofosbuvir/química , Sofosbuvir/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Sofosbuvir (SOF) is a nucleotide prodrug which has been used as a backbone for the clinical treatment of hepatitis C viral infection. Because sofosbuvir undergoes complex first pass metabolism, including metabolic activation to form its pharmacologically active triphosphate (GS-331007-TP) to inhibit the viral RNA polymerase in the liver, it is difficult to project the human dose for clinical evaluation based on preclinical data. Selecting an appropriate animal model for drug exposure in the target tissue is challenging due to differences in absorption, stability, hepatic uptake, and intracellular activation across species. Efficient liver delivery has been established in human liver following administration in a clinical trial of patients receiving sofosbuvir prior to liver transplantation. Using the clinical liver exposure as a benchmark, we assessed and compared the pharmacokinetic profile in mouse, rat, hamster, dog and monkey. Liver accumulation was also assessed in the PXB mouse model in which the liver is mostly populated with human hepatocytes. At human equivalent dose, the hepatic concentrations of GS-331007-TP in dog and PXB mouse were comparable to those observed in the human livers. In these species, high and sustained levels of GS-331007-TP were observed in both primary hepatocytes in vitro and the liver in vivo.
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Hígado/química , Hígado/metabolismo , Profármacos/metabolismo , Sofosbuvir/metabolismo , Animales , Perros , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Conformación Molecular , Profármacos/química , Profármacos/farmacocinética , Ratas , Ratas Sprague-Dawley , Sofosbuvir/química , Sofosbuvir/farmacocinéticaRESUMEN
AIMS: A newly emerged Human Coronavirus (HCoV) is reported two months ago in Wuhan, China (COVID-19). Until today >2700 deaths from the 80,000 confirmed cases reported mainly in China and 40 other countries. Human to human transmission is confirmed for COVID-19 by China a month ago. Based on the World Health Organization (WHO) reports, SARS HCoV is responsible for >8000 cases with confirmed 774 deaths. Additionally, MERS HCoV is responsible for 858 deaths out of about 2500 reported cases. The current study aims to test anti-HCV drugs against COVID-19 RNA dependent RNA polymerase (RdRp). MATERIALS AND METHODS: In this study, sequence analysis, modeling, and docking are used to build a model for Wuhan COVID-19 RdRp. Additionally, the newly emerged Wuhan HCoV RdRp model is targeted by anti-polymerase drugs, including the approved drugs Sofosbuvir and Ribavirin. KEY FINDINGS: The results suggest the effectiveness of Sofosbuvir, IDX-184, Ribavirin, and Remidisvir as potent drugs against the newly emerged HCoV disease. SIGNIFICANCE: The present study presents a perfect model for COVID-19 RdRp enabling its testing in silico against anti-polymerase drugs. Besides, the study presents some drugs that previously proved its efficiency against the newly emerged viral infection.
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Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/química , Betacoronavirus/enzimología , Infecciones por Coronavirus/tratamiento farmacológico , Guanosina Monofosfato/análogos & derivados , Neumonía Viral/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Ribavirina/química , Sofosbuvir/química , Proteínas Virales/antagonistas & inhibidores , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Alanina/química , Alanina/metabolismo , Alphacoronavirus/enzimología , Alphacoronavirus/genética , Secuencia de Aminoácidos , Antivirales/metabolismo , Betacoronavirus/genética , COVID-19 , Dominio Catalítico , Biología Computacional/métodos , Infecciones por Coronavirus/virología , Reposicionamiento de Medicamentos/métodos , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Neumonía Viral/virología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Ribavirina/metabolismo , SARS-CoV-2 , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sofosbuvir/metabolismo , Termodinámica , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
After a long period of interferon-and ribavirin-based therapy (IFN/RBV), a very fast evolution in the development of directly acting antivirals (DAAs) has now established a totally new paradigm for the treatment chronic HCV infection. An efficacy rate within the 95-100% interval, safer and more tolerable drugs, much shorter treatment duration and a quicker establishment of the sustained virological response (SVR) are among the most relevant properties of new DAAs as compared to former IFN/RBV therapies. The last wave of DAAs is also characterized by a lesser tendency to generate or being victim of drug-drug interactions. Nevertheless, since the circumstances in which patients are also recipients of other medications are rather frequent, individualization of treatment is advised in order to minimize the risk of drug-drug interactions of clinical relevance. Three two-drug regimens are available in Italy for the treatment of chronic HCV infection: sofosbuvir/velpatasvir (SOF/VEL), glecaprevir/pibrentasvir (GLE/PIB) and grazoprevir/elbasvir (GZP/RLB). Based on the officially released summary of product characteristics (SmPC) of these three co-formulated dual regimens, we performed a comparative analysis concerning the drug-drug interactions possibly affecting the DAA regimens. According to specific individual conditions, including co-morbidities, the choice of the most appropriate regimen must carefully take into account, among the different variables, the metabolic profile of both DAAs and concurrent medications. The differences among the three regimens offer the possibility to avoid the occurrence of clinically relevant drug-drug interactions in most circumstance.
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Antivirales/farmacología , Bencimidazoles/farmacología , Benzofuranos/farmacología , Carbamatos/farmacología , Hepatitis C Crónica/tratamiento farmacológico , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Imidazoles/farmacología , Pirrolidinas/farmacología , Quinoxalinas/farmacología , Sofosbuvir/farmacología , Sulfonamidas/farmacología , Antivirales/metabolismo , Antivirales/uso terapéutico , Bencimidazoles/metabolismo , Benzofuranos/metabolismo , Carbamatos/metabolismo , Combinación de Medicamentos , Interacciones Farmacológicas , Hepatitis C Crónica/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Humanos , Imidazoles/metabolismo , Interferón-alfa/uso terapéutico , Italia , Polifarmacia , Pirrolidinas/metabolismo , Quinoxalinas/metabolismo , Sofosbuvir/metabolismo , Sulfonamidas/metabolismoRESUMEN
BACKGROUND: While direct-acting antivirals have been approved for treating hepatitis C, the guidelines highlight the importance of considering potential drug-drug interactions between DAAs and concomitant medications. AIM: To assess comorbidity prevalence, concomitant medication use and potential drug-drug interactions between DAAs and concomitant medications for hepatitis C patients in Taiwan. METHODS: This cross-sectional study enrolled 822 patients from May to August 2016 in Taiwan. Patient demographics, comorbidities and concomitant medications were evaluated by physician surveys. RESULTS: A total of 709 (86.3%) patients had ≥1 comorbidity; the most prevalent comorbidity categories were diseases of the digestive system (40.1%), circulatory system (38.7%) and endocrine/nutritional/metabolic diseases (35.2%). Elderly patients had more comorbidities. A total of 622 (75.7%) patients received ≥1 concomitant medication; the average number of concomitant medications was 3.2. The most common concomitant medication classes were cardiovascular (34.4%), gastrointestinal (25.7%) and central nervous system drugs (22.7%). Among patients without cirrhosis or with compensated cirrhosis, contraindications were most prevalent with paritaprevir/ritonavir/ombitasvir plus dasabuvir, daclatasvir/asunaprevir and glecaprevir/pibrentasvir (13.3%, 6.0% and 5.4% respectively), and least prevalent with sofosbuvir, sofosbuvir/daclatasvir, sofosbuvir/ledipasvir and sofosbuvir/velpatasvir (0.8%, 1.3%, 1.4% and 2.1% respectively). Sofosbuvir-based regimens had no contraindications in patients with decompensated cirrhosis. CONCLUSION: Our population represented an elderly demographic, with a high prevalence of comorbidities and widespread use of concomitant medications. The potential drug-drug interactions between these concomitant medications and DAA regimens differed, with the fewest potential interactions with sofosbuvir-based regimens.
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Antivirales/uso terapéutico , Bencimidazoles/uso terapéutico , Fluorenos/uso terapéutico , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/epidemiología , Sofosbuvir/uso terapéutico , Uridina Monofosfato/análogos & derivados , Anciano , Antiinfecciosos/metabolismo , Antiinfecciosos/uso terapéutico , Antivirales/metabolismo , Bencimidazoles/metabolismo , Fármacos Cardiovasculares/metabolismo , Fármacos Cardiovasculares/uso terapéutico , Comorbilidad , Estudios Transversales , Interacciones Farmacológicas/fisiología , Femenino , Fluorenos/metabolismo , Hepatitis C Crónica/metabolismo , Humanos , Interferones/metabolismo , Interferones/uso terapéutico , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Sofosbuvir/metabolismo , Taiwán/epidemiología , Uridina Monofosfato/metabolismo , Uridina Monofosfato/uso terapéuticoAsunto(s)
Hepatitis C Crónica/tratamiento farmacológico , Inhibidores de Proteasas/uso terapéutico , Bencimidazoles/efectos adversos , Bencimidazoles/metabolismo , Bencimidazoles/uso terapéutico , Carbamatos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Fluorenos/efectos adversos , Fluorenos/metabolismo , Fluorenos/uso terapéutico , Humanos , Imidazoles/efectos adversos , Imidazoles/metabolismo , Imidazoles/uso terapéutico , Isoquinolinas/efectos adversos , Isoquinolinas/metabolismo , Isoquinolinas/uso terapéutico , Riñón/efectos de los fármacos , Riñón/fisiología , Inhibidores de Proteasas/efectos adversos , Inhibidores de Proteasas/metabolismo , Pirrolidinas , Sofosbuvir/efectos adversos , Sofosbuvir/metabolismo , Sofosbuvir/uso terapéutico , Sulfonamidas/efectos adversos , Sulfonamidas/metabolismo , Sulfonamidas/uso terapéutico , Valina/análogos & derivadosRESUMEN
Rifampin demonstrated dose-dependent relative induction between cytochrome P (CYP)3A and P-glycoprotein (P-gp), organic anion transporting polypeptides (OATPs), or CYP2C9; P-gp, OATP, and CYP2C9 induction was one drug-drug interaction (DDI) category lower than that observed for CYP3A across a wide range of pregnane X receptor (PXR) agonism. The objective of this study was to determine if these relationships could be utilized to predict transporter induction by other CYP3A inducers (rifabutin and carbamazepine) and of another P-gp substrate, sofosbuvir. Healthy subjects received sofosbuvir and a six-probe drug cassette before and after 300 mg q.d. rifabutin or 300 mg b.i.d. carbamazepine. Induction of P-gp, CYP2C9, and decreased sofosbuvir exposure were successfully predicted by observed CYP3A induction. Carbamazepine induction of OATP was underpredicted, likely due to reported additional non-PXR agonism. The results demonstrate that the effect of a PXR agonist on CYP3A can be leveraged to inform on induction liability for other primarily PXR-regulated P450s/transporters, allowing for prioritization of targeted DDI assessments during new drug development.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/agonistas , Carbamazepina/administración & dosificación , Citocromo P-450 CYP3A/biosíntesis , Inductores de las Enzimas del Citocromo P-450/administración & dosificación , Moduladores del Transporte de Membrana/administración & dosificación , Receptor X de Pregnano/agonistas , Rifabutina/administración & dosificación , Rifampin/administración & dosificación , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adolescente , Adulto , Biotransformación , Carbamazepina/efectos adversos , Simulación por Computador , Citocromo P-450 CYP2C9/biosíntesis , Inductores de las Enzimas del Citocromo P-450/efectos adversos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inducción Enzimática , Femenino , Voluntarios Sanos , Humanos , Masculino , Moduladores del Transporte de Membrana/efectos adversos , Persona de Mediana Edad , Modelos Biológicos , Transportadores de Anión Orgánico/agonistas , Transportadores de Anión Orgánico/metabolismo , Receptor X de Pregnano/metabolismo , Rifabutina/efectos adversos , Rifampin/efectos adversos , Medición de Riesgo , Sofosbuvir/metabolismo , Especificidad por Sustrato , Adulto JovenRESUMEN
Dengvaxia® (CTD-TDV), the only licensed tetravalent dengue vaccine by Sanofi Pasteur, was made available since 2015. However, administration of CTD-TDV, in general, has not received the prequalification recommendation from the World Health Organization. Having a universal antidengue agent for treatment will therefore beneficial. Accordingly, the development of nucleoside inhibitors specific to dengue viral polymerase that perturb dengue infection has been studied by many. Alternatively, we have used a marketed anti-HCV prodrug sofosbuvir to study its in silico and in vitro effects against dengue. As a result, the active metabolite of sofosbuvir (GS-461203) was predicted to bind to the catalytic motif (Gly-Asp-Asp) of dengue viral polymerase with binding affinity of -6.9 kcal/mol. Furthermore, sofosbuvir demonstrated excellent in vitro viral inhibition with an EC90 of 0.4 µm. In addition, this study demonstrated the requirement of specific liver enzymes to activate the prodrug into GS-461203 to exert its antidengue potential. All in all, sofosbuvir should be subjected to in-depth studies to provide information of its efficacy toward dengue and its lead potential as DENV polymerase inhibitor in human subjects. In conclusion, we have expended the potential of the clinically available drug sofosbuvir as treatment for dengue.
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Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Sofosbuvir/farmacología , Secuencias de Aminoácidos , Animales , Antivirales/química , Antivirales/metabolismo , Sitios de Unión , Dominio Catalítico , Línea Celular , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Virus del Dengue/enzimología , Humanos , Simulación del Acoplamiento Molecular , Nucleósidos/química , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacología , Unión Proteica , Sofosbuvir/química , Sofosbuvir/metabolismo , Termodinámica , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.
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Antivirales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sofosbuvir/metabolismo , Activación Metabólica , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Mutación Puntual , Especificidad por SustratoRESUMEN
Sofosbuvir metabolite, 2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206) was studied for the first time by surface enhanced Raman spectroscopy (SERS) using the paper-based SERS substrate. The quantification limit of PSI-6206 by SERS was found to be 13ngL-1 (R2 value=0.959, RSD=5.23%). For the structural and quantitative analysis of PSI-6206 in blood plasma, an interference-free HPLC-SERS method was developed and compared to HPLC-DAD and HPLC-MS methods. The SERS quantification of the drug by the paper substrate was 4 orders of magnitude more sensitive than that by the diode array detector. In addition, the SERS detection provided unique structural identification of the drug in blood plasma, similar to Mass spectroscopy detector. Due to the disposable nature of the SERS substrate, the new method does not suffer from the known "memory effect" which is known to lead to false positive identification in traditional HPLC-SERS methods. Therefore, the presented HPLC-paper SERS platform holds great potential for the sensitive and cost effective determination of drugs and their metabolites in biological fluids.
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Antivirales/sangre , Cromatografía Líquida de Alta Presión/métodos , Desoxiuridina/análogos & derivados , Sofosbuvir/sangre , Espectrometría Raman/métodos , Animales , Antivirales/metabolismo , Cromatografía Líquida de Alta Presión/instrumentación , Desoxiuridina/sangre , Desoxiuridina/metabolismo , Oro/química , Caballos , Técnicas In Vitro , Límite de Detección , Nanoestructuras/química , Estándares de Referencia , Sofosbuvir/metabolismo , Espectrometría Raman/instrumentaciónRESUMEN
In targeting the essential viral RNA-dependent RNA-polymerase (RdRp), nucleotide analogues play a major role in antiviral therapies. In the Flaviviridae family, the hepatitis C virus (HCV) can be eradicated from chronically infected patients using a combination of drugs which generally include the 2'-modified uridine analogue Sofosbuvir, delivered as nucleotide prodrug. Dengue and Zika viruses are emerging flaviviruses whose RdRp is closely related to that of HCV, yet no nucleoside drug has been clinically approved for these acute infections. We have purified dengue and Zika virus full-length NS5, the viral RdRps, and used them to assemble a stable binary complex made of NS5 and virus-specific RNA primer/templates. The complex was used to assess the selectivity of NS5 towards nucleotide analogues bearing modifications at the 2'-position. We show that dengue and Zika virus RdRps exhibit the same discrimination pattern: 2'-O-Me > 2'-C-Me-2'-F > 2'-C-Me nucleoside analogues, unlike HCV RdRp for which the presence of the 2'-F is beneficial rendering the discrimination pattern 2'-O-Me > 2'-C-Me ≥ 2'-C-Me-2'-F. Both 2'-C-Me and 2'-C-Me-2'-F analogues act as non-obligate RNA chain terminators. The dengue and Zika NS5 nucleotide selectivity towards 2'-modified NTPs mirrors potency of the corresponding analogues in infected cell cultures.
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Virus del Dengue/enzimología , Nucleótidos/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Virus Zika/enzimología , Antivirales/química , Antivirales/metabolismo , Humanos , Nucleótidos/química , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/aislamiento & purificación , Sofosbuvir/química , Sofosbuvir/metabolismo , Especificidad por Sustrato , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/aislamiento & purificaciónRESUMEN
Sofosbuvir is a direct acting antiviral medication used to treat Hepatitis C viral infection. The present study focuses on the degradation behavior of the drug under various stress conditions (hydrolysis, oxidative, thermal and photolytic) as per International Conference on Harmonization (ICH Q1A (R2)) guidelines. A high performance liquid chromatographic system (HPLC) was used to develop a selective, precise and accurate method for separating all the degradation products. The separation was achieved on a Sunfire™ C18 (150mm×4.6mm×5µm) stationary phase with a mobile phase of 10mM ammonium acetate (pH 5.0) buffer and acetonitrile in gradient elution mode. A quadrupole-time of flight mass analyzer equipped with an electrospray ionization technique was used to propose the structural information based on the MS/MS and accurate mass measurements. Seven degradation products were identified and characterised by LC-ESI-QTOF-MS/MS. In silico toxicity of the drug and its degradation products was determined using TOPKAT and DEREK toxicity prediction softwares. The proposed method was validated as per the ICH Q2 guidelines.