RESUMO
Molecular tests that detect and/or quantify HCV RNA are important in the diagnosis and management of patients with chronic hepatitis C (CHC) undergoing anti-viral therapy. The primary goal of anti-HCV therapy is to achieve a sustained virologic response (SVR) defined as "undetectable" Hepatitis C Virus (HCV) RNA in the serum or plasma at 12 to 24 weeks following the end of treatment.
Assuntos
Antivirais/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Animais , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/patologia , Humanos , Pessoa de Meia-Idade , Patologia Molecular , Resultado do TratamentoRESUMO
Hepatitis C virus (HCV) RNA viral load (VL) monitoring is a well-established diagnostic tool for the management of chronic hepatitis C patients. HCV RNA VL results are used to make treatment decisions with the goal of therapy to achieve an undetectable VL result. Therefore, a sensitive assay with high specificity in detecting and accurately quantifying HCV RNA across genotypes is critical. Additionally, a lower sample volume requirement is desirable for the laboratory and the patient. This study evaluated the performance characteristics of a second-generation real-time PCR assay, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2.0 (CAP/CTM HCV test, v2.0), designed with a novel dual-probe approach and an optimized automated extraction and amplification procedure. The new assay demonstrated a limit of detection and lower limit of quantification of 15 IU/ml across all HCV genotypes and was linear from 15 to 100,000,000 IU/ml with high accuracy (<0.2-log(10) difference) and precision (standard deviation of 0.04 to 0.22 log(10)). A specificity of 100% was demonstrated with 600 HCV-seronegative specimens without cross-reactivity or interference. Correlation to the Cobas AmpliPrep/Cobas TaqMan HCV test (version 1) was good (n = 412 genotype 1 to 6 samples, R(2) = 0.88; R(2) = 0.94 without 105 genotype 4 samples). Paired plasma and serum samples showed similar performance (n = 25, R(2) = 0.99). The sample input volume was reduced from 1 to 0.65 ml in the second version. The CAP/CTM HCV test, v2.0, demonstrated excellent performance and sensitivity across all HCV genotypes with a smaller sample volume. The new HCV RNA VL assay has performance characteristics that make it suitable for use with currently available direct-acting antiviral agents.
Assuntos
Hepacivirus/genética , Hepatite C/diagnóstico , Hepatite C/virologia , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Carga Viral , Genótipo , Humanos , Sondas de Ácido Nucleico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly4Ser)n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18- to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibody-resistant HIV-1 strains.
Assuntos
Anticorpos Biespecíficos/farmacologia , HIV-1/efeitos dos fármacos , Receptores CCR5/imunologia , Linhagem Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , HIV-1/imunologia , HumanosRESUMO
BACKGROUND: Massively parallel DNA sequencing of cell-free fetal DNA from maternal blood can detect fetal chromosomal abnormalities. Although existing algorithms focus on the detection of fetal trisomy 21 (T21), these same algorithms have difficulty detecting trisomy 18 (T18). METHODS: Blood samples were collected from 1014 patients at 13 US clinic locations before they underwent an invasive prenatal procedure. All samples were processed to plasma, and the DNA extracted from 119 samples underwent massively parallel DNA sequencing. Fifty-three sequenced samples came from women with an abnormal fetal karyotype. To minimize the intra- and interrun sequencing variation, we developed an optimized algorithm by using normalized chromosome values (NCVs) from the sequencing data on a training set of 71 samples with 26 abnormal karyotypes. The classification process was then evaluated on an independent test set of 48 samples with 27 abnormal karyotypes. RESULTS: Mapped sites for chromosomes of interest in the sequencing data from the training set were normalized individually by calculating the ratio of the number of sites on the specified chromosome to the number of sites observed on an optimized normalizing chromosome (or chromosome set). Threshold values for trisomy or sex chromosome classification were then established for all chromosomes of interest, and a classification schema was defined. Sequencing of the independent test set led to 100% correct classification of T21 (13 of 13) and T18 (8 of 8) samples. Other chromosomal abnormalities were also identified. CONCLUSION: Massively parallel sequencing is capable of detecting multiple fetal chromosomal abnormalities from maternal plasma when an optimized algorithm is used.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos/genética , DNA/genética , Feto , Diagnóstico Pré-Natal/métodos , Adolescente , Adulto , Algoritmos , Sistema Livre de Células , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 21/genética , DNA/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Gravidez , Análise de Sequência de DNA , Cromossomos Sexuais/genética , Trissomia/diagnóstico , Gêmeos , Adulto JovemRESUMO
Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used material for tumor molecular profiling, therapy selection, and prognostication. Tumor tissue enrichment by tissue dissection is highly recommended to generate quality data reproducibly for use in downstream assays, such as real-time PCR and next-generation sequencing. The aim of this study was to evaluate the performance of the automated tissue dissection tool AVENIO Millisect System compared with a manual dissection method using 18 FFPE tissue specimens. The study assessed performance of these two methods with paraffinized and deparaffinized sections at 5- and 10-µm thickness as well as at low (5% to 10%) and high (>50%) tumor content. In addition, compatibility with various nucleic acid and protein extraction methods was assessed. Overall, dissection by Millisect resulted in statistically significantly higher yields of nucleic acids and protein compared with manual dissection (P = 0.00524). In downstream analysis on a statistically nonpowered sample set, EGFR mutation testing by PCR led to highly concordant results, and next-generation sequencing testing yielded significantly higher allelic frequencies when tissue was dissected by Millisect compared with manual scraping, demonstrating noninferiority of the automated method. In summary, the AVENIO Millisect System may replace manual labor and support automation of FFPE tumor tissue workflows in clinical molecular laboratories with high testing volumes with adequate validation.
Assuntos
Dissecação/métodos , Fixadores/química , Formaldeído/química , Técnicas de Diagnóstico Molecular/métodos , Neoplasias/diagnóstico , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Confiabilidade dos Dados , Receptores ErbB/genética , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Pulmão , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Oncologia/métodos , Mutação , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos TestesRESUMO
In passaging experiments, we isolated HIV strains resistant to MAb3952, a chemokine (C-C motif) receptor 5 (CCR5) monoclonal antibody (MAb) that binds to the second extracellular domain (extracellular loop 2 [ECL-2]) of CCR5. MAb3952-resistant viruses remain CCR5-tropic and are cross-resistant to a second ECL-2-specific antibody. Surprisingly, MAb3952-resistant viruses were more susceptible to RoAb13, a CCR5 antibody binding to the N terminus of CCR5. Using CCR5 receptor mutants, we show that MAb3952-resistant virus strains preferentially use the N terminus of CCR5, while the wild-type viruses preferentially use ECL-2. We propose this switch in the CCR5 binding site as a novel mechanism of HIV resistance.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Epitopos/imunologia , Infecções por HIV/tratamento farmacológico , HIV/efeitos dos fármacos , Receptores CCR5/imunologia , Fármacos Anti-HIV/farmacologia , Sítios de Ligação/genética , Linhagem Celular Tumoral , Células Cultivadas , Farmacorresistência Viral/genética , Epitopos/química , Epitopos/genética , HIV/genética , Infecções por HIV/virologia , Humanos , Receptores CCR5/química , Receptores CCR5/genéticaRESUMO
The introduction of N-substituted pyrazoles in a new series of CCR5 antagonists was shown to substantially increase antiviral activity.
Assuntos
Fármacos Anti-HIV/química , Antagonistas dos Receptores CCR5 , Pirazóis/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Humanos , Pirazóis/síntese química , Pirazóis/farmacologia , Receptores CCR5/metabolismo , Relação Estrutura-AtividadeRESUMO
Replacement of a secondary amide with a piperidine or azetidine moiety in a series of CCR5 antagonists led to the discovery of compounds with increased intrinsic permeability. This effort led to the identification of a potent CCR5 antagonist which exhibited an improved in vivo pharmacokinetic profile.
Assuntos
Amidas/química , Compostos Aza/farmacologia , Antagonistas dos Receptores CCR5 , Compostos Aza/química , Compostos Aza/farmacocinética , Relação Estrutura-AtividadeRESUMO
Elaboration of our previously disclosed spiropiperidine template led to the development of a series of novel CCR5 antagonists. Results of SAR exploration and preliminary lead characterization are described.
Assuntos
Antagonistas dos Receptores CCR5 , Imidazolidinas/síntese química , Imidazolidinas/farmacologia , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Imidazolidinas/química , Imidazolidinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
The bicyclic 5-amino-3-azabicyclo[3.3.0]octanes were shown to be effective replacements for the 3-amino-8-azabicyclo[3.2.1]octane found in the CCR5 antagonist maraviroc.
Assuntos
Fármacos Anti-HIV/química , Compostos Azabicíclicos/química , Antagonistas dos Receptores CCR5 , Cicloexanos/química , Inibidores da Fusão de HIV/química , Triazóis/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Linhagem Celular Tumoral , Cicloexanos/síntese química , Cicloexanos/farmacologia , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/farmacologia , Humanos , Maraviroc , Modelos Químicos , Receptores CCR5/metabolismo , Triazóis/síntese química , Triazóis/farmacologiaRESUMO
Replacement of a secondary amide with an N-acyl or N-sulfonyl gem-disubstituted azacyle in a series of CCR5 antagonists led to the identification of compounds with excellent in vitro HIV antiviral activity and increased intrinsic membrane permeability.
Assuntos
Amidas/química , Fármacos Anti-HIV/química , Compostos Aza/química , Antagonistas dos Receptores CCR5 , Inibidores da Fusão de HIV/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Compostos Aza/síntese química , Compostos Aza/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/farmacologia , Humanos , Receptores CCR5/metabolismoRESUMO
The bicyclic 5-amino-3-azabicyclo[3.3.0]octanes were shown to be effective replacements for the conformationally restricted 4-aminopiperidine ring found in several series of CCR5 antagonists.
Assuntos
Antagonistas dos Receptores CCR5 , Avaliação Pré-Clínica de Medicamentos , Piperidinas/química , Modelos MolecularesRESUMO
Further investigation of the recently reported piperidine-4-yl-aminopyrimidine class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant viruses. Synthesis, structure-activity relationship (SAR), clearance data, and crystallographic evidence for the binding motif are discussed.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Pirimidinas/química , Pirimidinas/farmacologia , Fármacos Anti-HIV/síntese química , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , Humanos , Modelos Moleculares , Mutação , Piperidinas/síntese química , Piperidinas/química , Piperidinas/farmacologia , Pirimidinas/síntese química , Relação Estrutura-AtividadeRESUMO
An analysis of the binding motifs of known HIV-1 non-nucleoside reverse transcriptase inhibitors has led to discovery of novel piperidine-linked aminopyrimidine derivatives with broad activity against wild-type as well as drug-resistant mutant viruses. Notably, the series retains potency against the K103N/Y181C and Y188L mutants, among others. Thus, the N-benzyl compound 5k has a particularly attractive profile. Synthesis and SAR are presented and discussed, as well as crystal structures relating to the binding motifs.
Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Mutação , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Descoberta de Drogas , Farmacorresistência Viral/genética , HIV-1/genética , Modelos Moleculares , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
Starting with a high-throughput screening lead, a novel series of CCR5 antagonists was developed utilizing an information-based approach. Improvement of pharmacokinetic properties for the series was pursued by SAR exploration of the lead template. The synthesis, SAR and biological profiles of the series are described.
Assuntos
Fármacos Anti-HIV/química , Benzamidas/química , Antagonistas dos Receptores CCR5 , Inibidores da Fusão de HIV/química , Pirróis/química , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacocinética , Benzamidas/síntese química , Benzamidas/farmacologia , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/farmacocinética , Humanos , Microssomos Hepáticos/metabolismo , Pirróis/síntese química , Pirróis/farmacocinética , Ratos , Receptores CCR5/metabolismo , Relação Estrutura-AtividadeRESUMO
Replacement of the cyclic carbamate in our previously disclosed 1-oxa-3,9-diazaspiro[5.5]undecan-2-one template led to the discovery of two novel series of 3,9-diazaspiro[5.5]undecane and undeca-2-one CCR5 antagonists. The synthesis, SAR, and antiviral activities of these two series are described. One compound (32) was found to have attractive combination of antiviral potency, selectivity, and pharmacokinetic profile. The asymmetric synthesis of 32 was also accomplished and both enantiomers were equally potent.
Assuntos
Alcanos/síntese química , Antivirais/síntese química , Antagonistas dos Receptores CCR5 , Compostos de Espiro/síntese química , Administração Oral , Alcanos/farmacologia , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Disponibilidade Biológica , Descoberta de Drogas , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.
Assuntos
Antagonistas dos Receptores CCR5 , Piperidinas/química , Piperidinas/farmacologia , Receptores CCR5/metabolismo , Animais , Células CACO-2 , Cães , Haplorrinos , Humanos , Piperidinas/farmacocinética , Ratos , Compostos de Espiro/química , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The highest burden of disease from hepatitis C virus (HCV) is found in Southeast Asia, but our understanding of the epidemiology of infection in many heavily burdened countries is still limited. In particular, there is relatively little data on acute HCV infection, the outcome of which can be influenced by both viral and host genetics which differ within the region. We studied HCV genotype and IL28B gene polymorphism in a cohort of acute HCV-infected patients in Southern Vietnam alongside two other cohorts of chronic HCV-infected patients to better understand the epidemiology of HCV infection locally and inform the development of programs for therapy with the increasing availability of directly acting antiviral therapy (DAAs). METHODS: We analysed plasma samples from patients with acute and chronic HCV infection, including chronic HCV mono-infection and chronic Human Immunodeficiency Virus (HIV)-HCV coinfection, who enrolled in four epidemiological or clinical research studies. HCV infection was confirmed with RNA testing. The 5' UTR, core and NSB5 regions of HCV RNA positive samples were sequenced, and the genotype and subtype of the viral strains were determined. Host DNA from all HCV positive patients and age- and sex-matched non-HCV-infected control individuals were analysed for IL28B single nucleotide polymorphism (SNP) (rs12979860 and rs8099917). Geolocation of the patients were mapped using QGIS. RESULTS: 355 HCV antibody positive patients were analysed; 54.6% (194/355) and 46.4% (161/355) were acute and chronic infections, respectively. 50.4% (81/161) and 49.6.4% (80/161) of chronic infections had HCV mono-infection and HIV-HCV coinfection, respectively. 88.7% (315/355) and 10.1% (36/355) of the patients were from southern and central regions of Vietnam, respectively. 92.4% (328/355) of patients were HCV RNA positive, including 86.1% (167/194) acute and 100% (161/161) chronic infections. Genotype could be determined in 98.4% (322/328) patients. Genotypes 1 (56.5%; 182/322) and 6 (33.9%; 109/322) predominated. Genotype 1 including genotype 1a was significantly higher in HIV-HCV coinfected patients compared to acute HCV patients [43.8% (35/80) versus 20.5% (33/167)], (p = <0.001), while genotype 6 was significantly higher in chronic HCV mono-infected patients [(44.4% (36/81) versus 20.0% (16/80)] (p = < 0.004) compared to HIV-HCV coinfected patients. The prevalence of IL28B SNP (rs12979860) homozygous CC was 86.46% (83/96) in control individuals and was significantly higher in acutely-infected compared to chronically-infected patients [93.2 (82/88) versus 76.1% (35/46)] (p = < 0.005). CONCLUSION: HCV genotype 6 is highly prevalent in Vietnam and the high prevalence in treatment naïve chronic HCV patients may results from poor spontaneous clearance of acute HCV infection with genotype 6.
Assuntos
Coinfecção , Genótipo , Infecções por HIV , HIV-1/genética , Hepacivirus/genética , Hepatite C Crônica , RNA Viral/genética , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Coinfecção/epidemiologia , Coinfecção/genética , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/genética , Hepatite C Crônica/epidemiologia , Hepatite C Crônica/genética , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Prevalência , Vietnã/epidemiologiaRESUMO
We describe a novel strategy in which two inhibitors of HIV viral entry were incorporated into a single molecule. This bifunctional fusion inhibitor consists of an antibody blocking the binding of HIV to its co-receptor CCR5, and a covalently linked peptide which blocks envelope mediated virus-cell fusion. This novel bifunctional molecule is highly active on CCR5- and X4-tropic viruses in a single cycle assay and a reporter cell line with IC50 values of 0.03-0.05 nM. We demonstrated that both inhibitors contribute to the antiviral activity. In the natural host peripheral blood mononuclear cells (PBMC) the inhibition of CXCR4-tropic viruses is dependant on the co-expression of CCR5 and CXCR4 receptors. This bifunctional inhibitor may offer potential for improved pharmacokinetic parameters for a fusion inhibitor in humans and the combination of two active antiviral agents in one molecule may provide better durability in controlling the emergence of resistant viruses.
Assuntos
Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Receptores CCR5/metabolismo , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Antagonistas dos Receptores CCR5 , Linhagem Celular , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Receptores CCR5/imunologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Novel non-nucleoside inhibitors of HIV-RT that contain pyridazinone isosteres were prepared, and a series of triazolinones were found to be potent inhibitors of HIV replication. These compounds were active against several NNRTI-resistant virus strains. Pharmacokinetic studies indicated that inhibitor 7e has good bioavailability in rats. Several fragments of inhibitor 7c were prepared, and the binding of these compounds to HIV-RT was analyzed by surface plasmon resonance spectroscopy.